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Biotechnology Consultation
Note to the File
BNF No. 000098
Date: March 7, 2005
Subject: Glyphosate-tolerant cotton line MON 88913
Keywords: cotton, Gossypium hirsutum, glyphosate, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Agrobacterium sp. strain CP4, Roundup®, herbicide tolerant, CP4 EPSPS, MON 88913, MON-88913-8
In a submission dated May 27, 2004, Monsanto Company (Monsanto) submitted to FDA a safety and nutritional assessment of genetically engineered Roundup Ready® (glyphosate-tolerant) cotton line MON 88913. Monsanto provided additional information in support of this safety and nutritional assessment on November 8, and December 10, 2004. Monsanto concluded that food and feed derived from MON 88913 are as safe and nutritious as food and feed derived from conventional cotton.
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 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and prevents the synthesis of aromatic amino acids that are necessary for plant growth. Glyphosate-tolerant cotton line MON 88913 contains two cp4 epsps genes from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS enzyme. This enzyme has a reduced affinity for glyphosate when compared to the native plant EPSPS enzyme. As a result, cotton plants expressing the CP4 EPSPS enzyme are tolerant to glyphosate and survive spraying with glyphosate herbicide. Glyphosate-tolerant cotton line MON 88913 contains two cp4 epsps expression cassettes, each with different promoters, within a single insert. These cassettes are intended to provide increased expression of CP4 EPSPS in the cotton plant compared to existing glyphosate-tolerant cotton lines. The increased CP4 EPSPS expression will confer additional glyphosate tolerance at critical periods of plant development.
The parental cotton variety used in the transformation was Coker 312. Coker 312 is a commercial variety of upland cotton (Gossypium hirsutum L.).
Monsanto constructed the double border, binary plasmid vector PV-GHGT35 for the transformation of cotton variety Coker 312. The plasmid contains a single copy of two cp4 epsps expression cassettes within the T-DNA region. The T-DNA region of PV-GHGT35 was incorporated into the target cotton genome using Agrobacterium-mediated transformation. The T-DNA region of PV-GHGT35 contains the following elements.
| Elements of T-DNA region of PV-GHGT35 | Description |
|---|---|
| RB | Right border sequence derived from Agrobacterium tumefaciens |
| P-FMV/TSF1 | Chimeric promoter containing the Arabidopsis thaliana tsf1 gene promoter with enhancer sequences from the Figwort Mosaic virus 35S promoter |
| TSF1 (leader) | Leader from the Arabidopsis thaliana tsf1 gene encoding elongation factor EF-1alpha |
| TSF1 (intron) | Intron from the Arabidopsis thaliana tsf1 gene encoding elongation factor EF-1alpha |
| ctp2 | Chloroplast transit peptide from Arabidopsis thaliana derived from the Arabidopsis thaliana epsps gene. |
| cp4 epsps | 5-enolpyruvylshikimate-3-phosphate synthase enzyme gene derived from Agrobacterium sp. strain CP4 |
| E9 | DNA sequence derived from Pisum sativum containing the 3' nontranslated region of the pea ribulose-1,5 bisphosphate carboxylase, small subunit (rbc) E9 gene. |
| P-35S/ACT8 | Chimeric promoter containing the promoter of the ACT8 gene of Arabidopsis thaliana combined with the enhancer sequences of the Cauliflower Mosaic Virus 35S promoter. |
| ACT8 (leader) | Leader sequences from the act8 gene of Arabidopsis thaliana |
| ACT8 (intron, exon) | Intron and flanking exon sequence from the act8 gene of Arabidopsis thaliana |
| ctp2 | Chloroplast transit peptide from Arabidopsis thaliana derived from the Arabidopsis thaliana epsps gene. |
| cp4 epsps | 5-enolpyruvylshikimate-3-phosphate synthase enzyme gene derived from Agrobacterium sp. strain CP4 |
| E9 | DNA sequence derived from Pisum sativum containing the 3' nontranslated region of the pea ribulose-1,5 bisphosphate carboxylase, small subunit (rbc) E9 gene. |
| LB | Left border sequence derived from Agrobacterium tumefaciens. |
Plasmid PV-GHGT35 also contains several genes from the plasmid backbone necessary for maintenance and selection of the plasmid that are not ultimately incorporated into the plant genome. Plasmid PV-GHGT35 contains both vegetative and bacterial origins of replication that allow replication of the plasmid in both A. tumefaciens and Escherichia coli. The plasmid contains the aad gene encoding the Tn7 adenyltransferase that provides resistance to spectinomycin and streptomycin. The plasmid also contains a sequence, known as rop, which represses the formation of RNA primer thereby allowing maintenance and copy number control of the plasmid in E. coli.
Monsanto reports that the transformation resulted in the introduction of a single copy of the T-DNA region containing the two cp4 epsps expression cassettes into the cotton genome.
Monsanto used Southern blot analysis to characterize the DNA introduced in MON 88913. Based on this analysis, Monsanto concludes that event MON 88913 contains a single copy of the two cp4 epsps expression cassettes with each component of the cassettes intact. PCR analysis confirmed the organization of the elements within the DNA insert of MON 88913. Monsanto also concludes that event MON 88913 does not contain plasmid backbone sequences.
To ascertain whether the inserted DNA in MON 88913 was stably integrated into the genome, Monsanto performed Southern blot analyses on genomic DNA from generational descendants (R1, R2, R3, R4, R5) of the original transformant. Monsanto states that these analyses demonstrate that the inserted DNA, containing the full-length cp4 epsps expression cassettes, was stably integrated at a single locus in all progeny.
To examine the inheritance of the glyphosate tolerance trait, Monsanto conducted crosses using conventional breeding techniques. Chi-square analysis indicates that the introduced trait (glyphosate tolerance) was stably inherited through multiple generations and segregated according to a Mendelian inheritance pattern.
Native plant EPSPS enzymes are involved in the biosynthesis of aromatic amino acids that are necessary for growth and development of the plant. The CP4 EPSPS protein is similar in structure and function to native plant EPSPS enzymes, but has a much reduced affinity for glyphosate. The CP4 EPSPS expressed in cotton MON 88913 is a 47.6 kDa protein consisting of a single polypeptide of 455 amino acids. Monsanto used immunoblot analysis and densitometry, matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry, N-terminal sequence analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry, CP4 EPSPS enzyme assay, and glycosylation analysis to characterize plant-produced CP4 EPSPS and to confirm its identity.
Monsanto measured levels of CP4 EPSPS protein in MON 88913 young leaf, overseason leaf (OSL), root, seed, and pollen tissues with a validated enzyme-linked immunosorbent assay (ELISA). Monsanto reports that tissue samples were collected from MON 88913 produced in replicated field trials across four U.S. field locations during 2002. Monsanto states that three OSL samples were taken at different time points throughout the growing season (OSL1, OSL2, OSL3). Monsanto reports that mean levels (dry weight basis) of the CP4 EPSPS protein for young leaf, OSL1, OSL2, OSL3, root and seed tissues of MON 88913 were 970, 1400, 690, 630, 99, and 340 μg/g, respectively. Monsanto reports that the mean level of CP4 EPSPS protein in pollen was 4.0 μg/g (fresh weight).
Monsanto has estimated human and farm animal dietary exposure to CP4 EPSPS derived from MON 88913. Monsanto notes that the primary form of cottonseed consumed by humans is processed cottonseed oil. Monsanto states that farm animals may be exposed to the CP4 EPSPS protein through whole cottonseed, cottonseed meal, hulls, and other processing byproducts derived from MON 88913.
Based on dietary consumption data and estimates of the concentration of CP4 EPSPS in cottonseed oil and feed, Monsanto has estimated a daily dietary exposure (DDE) for adult humans and dairy cows, assuming that 100% of cottonseed, or products derived from cottonseed, are derived from event MON 88913. Monsanto estimates human DDE for CP4 EPSPS derived from MON 88913 in cottonseed oil to be 1.26 x 10-4 milligrams(mg)/kilogram(kg) body weight(wt)/day. Monsanto estimates a dairy cow's DDE to CP4 EPSPS derived from MON 88913 in feed to be 1.80 x 10-3 mg/kg body wt/day, assuming cottonseed intake of 5.3 grams/kg body wt/day.
Monsanto compared the amino acid sequence of CP4 EPSPS in cotton event MON 88913 to amino acid sequences in a collection of databases comprising all known protein sequences. Monsanto reports that no biologically relevant sequence similarities between the CP4 EPSPS protein and known toxins were observed.
Monsanto describes an acute toxicity study conducted in mice where male and female mice were dosed by gavage with up to 572 mg/kg body weight of CP4 EPSPS protein produced in E. coli.1 Monsanto reports that no adverse events were observed at any dose level.
Monsanto provided 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 evaluated during Monsanto's consultations with FDA on other glyphosate-tolerant crops.
Monsanto describes its assessment of the allergenic potential of the CP4 EPSPS protein. Monsanto states that the CP4 EPSPS protein was derived from Agrobacterium sp. strain CP4 and that there have been no reports of allergy to Agrobacterium species. Monsanto screened the amino acid sequence of CP4 EPSPS in cotton event MON 88913 for similarity to known allergens using a database containing all known allergen and gliadin protein sequences. Monsanto concluded that CP4 EPSPS in cotton event MON 88913 does not share structurally relevant or immunologically significant identity with any known allergens or gliadins.
Monsanto discusses studies performed to assess the in vitro digestibility of CP4 EPSPS. These studies used the CP4 EPSPS protein expressed in E. coli.1 In a study conducted by Harrison et al. (1996), the CP4 EPSPS protein was exposed to simulated gastric and intestinal fluids that were prepared according to the U.S. Pharmacopoeia (1990). The degradation of the protein was assessed by western blot analysis. The half-life of the CP4 EPSPS protein was reported to be less than 15 seconds in the simulated gastric fluid (SGF) and less than ten minutes in the simulated intestinal fluid (Harrison et al., 1996).
In subsequent studies, the in vitro digestibility of CP4 EPSPS was assessed in simulated gastric fluid, and the degradation of CP4 EPSPS in SGF was assessed by the SDS-PAGE colloidal blue gel staining method, western blot analysis, and EPSPS enzymatic 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 EPSPS activity was reduced to less than 10% of the initial activity within 15 seconds of exposure to SGF.
Food uses of Gossypium species cotton are limited due to the presence of natural toxicants present in cottonseed: gossypol and cyclopropenoid fatty acids. Therefore, only highly refined products made from cottonseed are suitable for human consumption. These products include cottonseed oil and, to a lesser extent, cottonseed linters. Refined cottonseed oil is a premium-quality oil used for a variety of food uses, including frying oil, salad and cooking oil, salad dressing, mayonnaise, shortening, margarine and packing oil. Linters are the short fibers that remain on the cottonseed after ginning. These linters are processed into pure cellulose for use in a variety of food and industrial products. Some linter fiber is used in food casings for bologna and sausages and in salad dressing to improve viscosity. Monsanto reports that cottonseed oil and linters are not expected to contain any detectable protein.
Whole cottonseed, cottonseed meal, crude cottonseed oil, hulls and gin trash are used in animal feeds for cattle, sheep, goats, horses, poultry, swine, fish and shrimp. Meal is typically sold as a 38-44% protein product or a 35% protein cottonseed cake. Cottonseed hulls are a byproduct of the oil extraction process and are used as a source of fiber for cattle to aid in digestion. Nearly all of the cottonseed produced in the U.S. is used for feed in various forms. Approximately 50-60% of the cottonseed is processed into oil, meal, and hulls. The remaining cottonseed is fed to cattle as whole cottonseed.
To assess whether glyphosate-tolerant cotton MON 88913 is as safe and nutritious as conventional cotton varieties that are currently consumed, Monsanto compared the composition of MON 88913 to the composition of a non-transgenic negative segregant variety MON 88913(-) and several commercial conventional varieties for use as reference material grown alongside MON 88913 at various field sites. MON 88913(-) is described as having a genetic background representative of MON 88913 without the CP4 EPSPS transformation. Compositional analysis data from the commercial varieties were used for calculating 99% tolerance intervals that were expected to contain, with 95% confidence, 99% of the values contained in the population of conventional cotton. Monsanto provides analytical results and comparisons of components of whole cottonseed, cottonseed meal, and cottonseed oil from MON 88913 with comparable products from MON 88913(-) and commercial reference varieties.
Analytical results are expressed as means and ranges from individual test sites and all sites combined. For purposes of statistical analysis and comparison to literature values, the whole cottonseed values for proximates (except moisture), fiber gossypol, minerals and vitamin E were converted from a fresh weight basis to a dry weight basis. Analytical results for amino acids and fatty acids were expressed as a percent of total amino acids and total fatty acids, respectively2. Monsanto conducted a mixed model analysis of variance statistical analysis on the converted values and statistically significant differences between MON 88913 and MON 88913(-) were declared at p ≤ 0.05.
For compositional analysis of whole delinted cottonseed, Monsanto evaluated MON 88913, MON 88913(-), and commercial cottonseed varieties grown under replicated conditions at four sites in cotton-producing regions in the U.S. in 2002. At each location, MON 88913, MON 88913(-) and four different commercial varieties were grown. A total of 16 different commercial varieties produced alongside MON 88913 were used as reference material for generating 99% tolerance intervals for composition of commercial conventional cottonseed. The cottonseed was harvested, ginned and acid-delinted prior to analysis.
Monsanto analyzed the samples of whole, acid-delinted cottonseed for the following components:
Monsanto conducted a combined statistical analysis of the analytical results from all of the field trials and found no statistically significant differences between MON 88913 and MON 88913(-), except for the amino acids phenylalanine and tryptophan when presented as a percent of total amino acids, the fatty acids oleic acid and linoleic acid when presented as a percent of total fatty acids, moisture, and the mineral manganese.3 Phenylalanine, linoleic acid and manganese mean levels were higher (p ≤ 0.05) for MON 88913 cottonseed than for MON 88913(-) cottonseed. Tryptophan and oleic acid mean levels in MON 88913 were lower (p ≤ 0.05) than that in MON 88913(-). These values were not outside the range of values for these components Monsanto measured in commercial cotton varieties. For comparisons in which MON 88913 was statistically different from MON 88913(-), Monsanto notes that the range of values for MON 88913 were within the 99% tolerance interval for the commercial conventional varieties, with the exception of moisture.4 The range of values for moisture in MON 88913 cottonseed, however did fall within the range of published values for conventional cottonseed. Therefore, Monsanto concludes that these differences are unlikely to be biologically meaningful.
For compositional analysis of cottonseed meal and cottonseed oil, Monsanto evaluated processed fractions of MON 88913, MON 88913(-) and commercial cotton varieties grown under replicated conditions at two sites in the U.S. in 2002. Two additional commercial reference varieties were grown at a third site. A total of six commercial varieties, four produced alongside MON 88913 were used as the reference material. Samples from the whole, ginned and mechanically-delinted cottonseed were analyzed to confirm that the cottonseed used for the production of oil and meal was compositionally equivalent to the control. The cottonseed was then processed into refined, bleached and deodorized cottonseed oil and raw, untoasted cottonseed meal. Raw cottonseed meal is described as the dehulled, flaked cottonseed that remains after solvent extraction (to remove oil) and air drying to remove residual solvent.
Monsanto analyzed the whole mechanically-delinted cottonseed (used in the production of cottonseed oil and meal) for proximates, ADF and NDF, crude fiber, TDF, amino acids, fatty acids, cyclopropenoid fatty acids, vitamin E, minerals, and gossypol. Monsanto conducted a combined statistical analysis of data from two field sites and detected no statistically significant differences except for the level of phenylalanine, which was found to be greater (p ≤ 0.05) in MON 88913 compared to MON 88913(-). However, the mean reported for phenylalanine was found to be within the range of literature values for cottonseed and within Monsanto's commercial range and calculated 99% tolerance interval for commercial conventional varieties.
Monsanto conducted analyses on the raw cottonseed meal to measure:
Monsanto reported no statistically significant differences in the mean levels of the analytes measured in cottonseed meal except for total gossypol in MON 88913 which was found to be less than (p ≤ 0.05) that in MON 88913(-). Monsanto notes that the range of values reported for gossypol in MON 88913 was found to be within the range of literature values for cottonseed meal and within Monsanto's commercial range and calculated 99% tolerance interval for commercial conventional varieties.
Refined, deodorized, cottonseed oil was analyzed for the following components:
Monsanto noted that in cottonseed oil, statistically significant differences were detected in the levels of the fatty acids myristic acid and behenic acid, which were both lower (p ≤ 0.05) in MON 88913 compared to MON 88913(-). However, Monsanto notes that the range of values for both myristic acid and behenic acid in MON 88913 were found to be within the range of published values for cottonseed oil, and/or within the range and 99% tolerance interval for commercial conventional varieties.
Monsanto concludes that glyphosate-tolerant cotton line MON 88913 is not materially different in terms of safety, composition, or any other relevant parameter from cotton now grown, marketed, and consumed. At this time, based on Monsanto's data and information, the agency considers Monsanto's consultation on glyphosate-tolerant cotton line MON 88913 to be complete.
/s/
Richard Bonnette
(1)In confirming the identity of the introduced CP4 EPSPS protein, Monsanto stated that CP4 EPSPS derived from event MON 88913 was found to be equivalent to CP4 EPSPS derived from E.coli.
(2)Monsanto also provided analytical values for amino acids and fatty acids in cottonseed expressed as a percentage of dry weight.
(3)The notifiers 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.
(4)FDA notes that the combined-site ranges of MON 88913 values for oleic acid and linoleic acid, expressed as a percentage of fatty acids, moisture (expressed on a fresh weight basis), and manganese, phenylalanine, and tryptophan expressed on a dry weight basis, were within the range of values reported for acid-delinted cottonseed in the International Life Sciences Institute (ILSI) Crop Composition Database (version 2.0, released April 5, 2004, available at www.cropcomposition.org). FDA notes that all MON 88913 values for amino acids expressed on a dry weight basis were within the range of values for acid-delinted cottonseed in the ILSI Crop Composition Database.
