Biotechnology Consultation Note to the File BNF No. 000135
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Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000135
April 19, 2013
Dicamba- and Glufosinate-Tolerant MON 88701 Cotton
Keywords: Cotton, Gossypium sp., dicamba mono-oxygenase, phosphinothricin N-acetyltransferase, MON 88701, Monsanto, herbicide tolerant, Stenotrophomonas maltophilia, Streptomyces hygroscopicus, OECD Unique Identifier MON-887Ø1-3
This document summarizes our evaluation of biotechnology notification file (BNF) No. 000135. In a submission dated April 6, 2012, Monsanto Company (Monsanto) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of genetically engineered MON 88701 cotton, which is tolerant to the herbicides dicamba and glufosinate. Monsanto provided additional information on May 22, July 18, and September 10, 2012. FDA evaluated the information in Monsanto’s submissions to ensure that regulatory and safety issues regarding the human food and animal feed from the new plant variety have been resolved prior to commercial distribution.
In our evaluation of BNF 000135, 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 all the information provided in the final consultation.
The intended effect of the modification in MON 88701 cotton is to produce dicamba- and glufosinate-tolerant cotton varieties.1 Monsanto states that the parental cotton variety was transformed with the codon optimized coding sequence of the dmo gene from Stenotrophomonas maltophilia and the coding sequence of the bar gene from Streptomyces hygroscopicus. The dmo gene encodes a dicamba mono-oxygenase protein (DMO), which confers tolerance to dicamba, and the bar gene encodes a phosphinothricin N-acetyltransferase protein (PAT (bar)), which confers tolerance to glufosinate.
The purpose of this evaluation is to assess whether Monsanto 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 Federal Insecticide, Fungicide, and Rodenticide Act and the FD&C Act. Under EPA regulations, the herbicides, metabolic by-products, and residues in MON 88701 cotton that result from detoxification of applied herbicides by the expression products of MON 88701 are considered pesticidal substances. Therefore, Monsanto plans to submit a regulatory submission to EPA for the expanded use of dicamba on MON 88701 cotton. Monsanto states that it will not pursue any changes in the established tolerances for the use of glufosinate on MON 88701 cotton.
Genetic Modification and Characterization
Monsanto transformed the non-transgenic conventional cotton variety Coker 130 to develop MON 88701 cotton.
Transformation Vector and Method
Monsanto states that MON 88701 cotton was developed through Agrobacterium tumefaciens-mediated transformation of Coker 130 hypocotyls with plasmid vector PV-GHHT6997. PV-GHHT6997 contains one T-DNA containing the dmo and bar expression cassettes. The DMO expression cassette consists of the following elements:
- the PC1SV promoter from the peanut chlorotic streak caulimovirus;
- the leader from the 5' untranslated region of the Tobacco Etch virus;
- the targeting sequence of the ShkG gene of Arabidopsis thaliana encoding the EPSPS chloroplast transit peptide (CTP2), which directs transport of the DMO protein to the chloroplast in MON 88701 cotton;
- the dmo coding sequence;2
- the 3' untranslated region of the E6 gene of Gossypium barbadense, which directs polyadenylation of the mRNA.
The bar expression cassette contains the following elements:
- the cauliflower mosaic virus e35S promoter containing a duplicated enhancer region;
- the leader from the 5' untranslated region of the Petunia hybrida DnaK gene;3
- the bar coding sequence from S. hygroscopicus;
- the 3' untranslated region of the A. tumefaciens nopaline synthase gene, which directs polyadenylation of the mRNA.
The T-DNA is delineated by left and right border regions, which facilitate transformation.
Monsanto selected the transformed plants with glufosinate to inhibit the growth of untransformed cells. R0 and R1 plants were screened for the presence of T-DNA and were evaluated for tolerance to dicamba and glufosinate. R1 plants homozygous for dmo and bar were self-pollinated to give rise to R2 plants.
Characterization, Inheritance, and Stability of the Introduced DNA
Monsanto characterized the introduced DNA, using Southern blot hybridization, polymerase chain reactions (PCR), and DNA sequence analyses. Southern blot analyses showed that MON 88701 cotton contains a single copy of the T-DNA inserted at a single locus in the genome and lacks any detectable plasmid backbone sequences.
According to Monsanto, PCR and DNA sequence analyses: 1) complemented the Southern blot analyses; 2) determined the complete DNA sequence of the T-DNA and flanking sequences; 3) confirmed the predicted organization of the genetic elements within the T-DNA, and 4) confirmed that each genetic element (except for the border regions) in the T-DNA is intact and that the sequence of the T-DNA is identical to the corresponding sequence in PV-GHHT6997.
During the development of MON 88701 cotton, Monsanto recorded segregation data to assess the stability of the introduced DNA in the genome and the heritability of the introduced DNA across generations. Southern blot analyses of genomic DNA from MON 88701 plants showed that the inserted DNA was stably maintained through five generations of breeding, confirming that the T-DNA is stably integrated into the genome. Chi-square analysis of the segregation data from three generations showed that the introduced DNA is inherited according to a Mendelian inheritance pattern of a single gene over multiple generations.
Monsanto conducted bioinformatic analyses on the six potential open reading frames (ORFs) in the inserted DNA and flanking DNA sequences. Based on these analyses, Monsanto concludes that even in the highly unlikely event of translation of these ORFs, the putative polypeptides are unlikely to be allergenic, toxic, or biologically active.
Identity, Function, and Characterization
DMO is a non-heme iron oxygenase that transports electrons from ferredoxin to the non-heme iron domain. The active enzyme is a trimer comprised of a reductase, a ferredoxin, and a terminal oxygenase. Diverse groups of organisms ranging from bacteria to plants produce oxygenases that humans and animals consume.
The dmo coding sequence within MON 88701 cotton produces a precursor protein consisting of the MON 88701 protein and CTP2. CTP2 improves targeting of the precursor protein to the chloroplast and is later removed to produce the full-length protein. MON 88701 cotton produces a form of DMO in which a fragment of CTP2 remains.
MON 88701 DMO has a sequence identical to wild type DMO protein from S. maltophilia with two exceptions: 1) MON 88701 DMO has nine amino acids on its N-terminus derived from CTP2, and 2) MON 88701 DMO has an additional leucine at position two. These amino acid differences are not expected to affect the structure, activity, or specificity of MON 88701 DMO because the N-terminus and position two are sterically distant from the protein’s catalytic site.
Monsanto assessed whether DMO can metabolize endogenous substrates present in MON 88701 cotton. Monsanto conducted in vitro experiments using a purified DMO, produced in Escherichia coli. This purified DMO contains an N-terminal tag containing histidine residues that was used in purifying the protein. Beyond this histidine-containing tag, this purified DMO is identical to DMO from S. maltophilia. The potential metabolic activity of DMO was tested on a set of substrates (o-anisic, vanillic, syringic, ferulic, and sinapic acids) endogenous to cotton with structural similarity to dicamba. Dicamba was used as a positive control to demonstrate that the assay system was functional. The purified DMO did not metabolize any of the tested substrates in the assay. Therefore, Monsanto concludes that MON 88701 DMO is specific for dicamba.
Monsanto also assessed whether MON 88701 DMO has the same specificity as the purified DMO used in the in vitro experiments described above. Monsanto purified an E. coli-produced MON 88701 DMO confirmed to be biochemically, structurally, and functionally equivalent to MON 88701 DMO produced in cotton.4 Monsanto tested the potential metabolic activity of E. coli-produced MON 88701 DMO with o-anisic acid (the endogenous compound with the greatest structural similarity to dicamba). E. coli-produced MON 88701 DMO did not metabolize o-anisic acid. Based on these results, Monsanto concludes that DMO, including MON 88701 DMO, is specific for dicamba.
MON 88701 DMO Expression Level
Monsanto measured MON 88701 DMO levels using an enzyme-linked immunosorbent assay (ELISA). MON 88701 cottonseed, pollen, root, and over-season leaf (OSL-1 through OSL-4) from eight field sites were analyzed.5 The levels of MON 88701 DMO across the tissue types ranged from below the limit of detection to 410 micrograms per gram dry weight (μg/g DW). The mean DMO concentrations (in μg/g DW) in MON 88701 cotton were 180 to 240 for leaf, 43 for root, and 21 for seed, and 14 μg/g fresh weight for pollen.6
Safety of the Donor Organism
Monsanto states that the dmo gene used to develop MON 88701 cotton is derived from S. maltophilia. S. maltophilia is an aerobic, Gram-negative, environmentally ubiquitous bacterium found in aquatic environments, household environments, soil, and plants. According to Monsanto, humans are exposed to S. maltophilia through consumption of ready-to-eat salads, vegetables, frozen fish, milk, and poultry. Published reports show that S. maltophilia is found in healthy individuals and does not cause harmful effects. The published literature also shows that S. maltophilia strains reside in the transient flora of hospitalized patients as commensal organisms and, similar to the indigenous bacteria of the gastrointestinal tract, can be opportunistic pathogens. Published reports suggest that human infections with S. maltophilia are rare and occur almost exclusively in hospital settings. S. maltophilia has not been reported to be a source of allergens. Based on this information, Monsanto concludes that S. maltophilia is a safe donor organism.
Assessment of Potential for Allergenicity and Toxicity
Monsanto states that the only human foods currently produced from cottonseed are refined, bleached, and deodorized (RBD) oil and linters, both of which contain negligible concentrations of protein. Nonetheless, Monsanto assessed the potential for allergenicity and toxicity of MON 88701 DMO.
To assess the potential allergenicity of MON 88701 DMO, Monsanto used the FASTA algorithm to compare the MON 88701 DMO amino acid sequence to amino acid sequences in the publicly available allergen, gliadin, and glutenin protein sequence database (AD_2011 database, Food Allergen Research and Resource Program, release date: February 18, 2011). According to Monsanto, no relevant sequence similarities (i.e. sequence alignments displaying an E-score of less than or equal to 1 x 10-5) were detected between the amino acid sequence of MON 88701 DMO and sequences in the AD_2011 database. Furthermore, no sequence alignment met or exceeded the threshold of 35% identity over 80 amino acids. Monsanto also performed an eight amino acid sliding window search to detect short linear polypeptide matches to known or suspected allergens in the AD_2011 database. No eight contiguous amino acids were detected when the MON 88701 DMO amino acid sequence was compared to sequences in the AD_2011 database. Monsanto states that the results of these analyses show that there are no structurally or immunologically relevant similarities between the amino acid sequence of MON 88701 DMO and sequences of known allergens, gliadins, and glutenins.
Monsanto tested the digestibility and heat stability of E. coli-produced MON 88701 DMO. Monsanto states that MON 88701 DMO is rapidly degraded in vitro in simulated gastric fluid and in simulated intestinal fluid. Additionally, in vitro analyses show that MON 88701 DMO remains intact, but is not catalytically active at temperatures of 55°C and above (commonly used during standard cotton processing). Monsanto concludes that in the unlikely event that cottonseed oil or linters would contain protein, MON 88701 DMO protein, if consumed, would not be catalytically active.
Monsanto used the FASTA algorithm to compare the MON 88701 DMO amino acid sequence to amino acid sequences in the toxin database, obtained as a subset of protein sequences from GenBank, which it refers to as the TOX_2011 database. The TOX_2011 database contains amino acid sequences of proteins that may be harmful to human and animal health. According to Monsanto, the results of the sequence comparisons found no relevant alignments (i.e., alignments displaying an E-score of less than or equal to 1 x 10-5) between the MON 88701 DMO amino acid sequence and sequences in the TOX_2011 database. Monsanto thus concludes that no sequence similarity exists between MON 88701 DMO and any known protein toxins or other biologically active proteins that would be harmful to human or animal health.
Monsanto conducted an acute oral toxicity study to further assess the potential for toxicity of MON 88701 DMO. Mice (10 male and 10 female) were given a single dose of 283 milligram per kilogram body weight (mg/kg bw) MON 88701 DMO by oral gavage or a comparable level of bovine serum albumin. This level was chosen based on margin of exposure and solubility of the protein. According to Monsanto, no treatment-related effects on survival, clinical observations, body weight gain, food consumption, or gross pathology were observed after 14 days in mice administered MON 88701 DMO .
Based on the results of the safety studies discussed above, Monsanto concludes that MON 88701 DMO does not pose a significant health risk to humans or animals.
Identity, Function, and Characterization
PAT (bar) is an acetyltransferase that acetylates the free amine group of L-phosphinothricin (a form of glufosinate) to produce the inactive metabolite N-acetyl glufosinate. PAT proteins have been isolated from two species of Streptomyces. The PAT protein isolated from S. hygroscopicus is encoded by the bar gene, and the PAT protein isolated from Streptomyces viridochromogenes is encoded by the pat gene. Monsanto states that the PAT protein produced in MON 88701 cotton is encoded by the bar gene, and is thus referred to as PAT (bar). Monsanto states that PAT (bar) is identical to the wild type PAT protein produced in S. hygroscopicus. PAT (bar) is analogous to PAT proteins produced in numerous commercially available glufosinate-tolerant plant varieties.
Monsanto conducted in vitro competition assays to assess whether PAT (bar) can metabolize substrates other than glufosinate. Addition of high concentrations of amino acids (including L-glutamate, a glufosinate analogue) did not inhibit glufosinate acetylation by PAT (bar). The results show that PAT (bar) has more than 30-fold higher affinity towards L-phosphinothricin over alternative substrates tested in the assay. Based on these data, Monsanto concludes that PAT (bar) is highly specific for L-phosphinothricin and is unlikely to affect the metabolism of MON 88701 cotton.
PAT (bar) Expression Level
Monsanto measured PAT (bar) levels in MON 88701 cotton using an ELISA.7 MON 88701 cottonseed, pollen, root, and OSL-1 through OSL-4 from eight field sites were analyzed. The levels of PAT (bar) across the tissue types ranged from below the limit of quantitation to 10 μg/g DW. The mean PAT (bar) levels (μg/g DW) were 6.6 for seed, 3.2 to 6.4 for leaf, 1.8 for root, and 0.56 μg/g FW for pollen.
Safety of the Donor Organism
Monsanto states that the bar gene introduced into the parental cotton variety is derived from S. hygroscopicus. S. hygroscopicus is a saprophytic, soil-borne bacterium that is widespread in the environment. The published literature shows that human exposure to S. hygroscopicus is common, and the organism is not considered pathogenic to plants, humans, or other animals. The history of safe use of S. hygroscopicus is documented in the literature, and no safety or allergenicity issues have been identified during evaluations of several glufosinate-tolerant events. Monsanto thus concludes that S. hygroscopicus is a safe donor organism.
The analytical methods that were used to characterize DMO were also used to demonstrate that E. coli-produced PAT (bar) is equivalent to that produced in MON 88701 cotton.
Assessment of Potential for Allergenicity and Toxicity
Monsanto states that the safety of PAT (bar) expressed in genetically engineered crops has been extensively assessed and that no concerns have been identified. Monsanto cites a published study demonstrating the safety of the donor organism, lack of amino acid sequence homology to known allergens, rapid degradation in gastric and intestinal fluids, and loss of functional activity following heat treatment. The results of bioinformatic analyses show that PAT (bar) lacks structurally and immunologically relevant sequence similarity to known allergens, gliadins, and glutenins. The results of in vitro analyses show that PAT (bar) is rapidly degraded in vitro in simulated gastric fluid and in simulated intestinal fluid. The results of in vitro analyses show that PAT (bar) is catalytically inactivated at temperatures of 75°C and above (commonly used during standard cotton processing); therefore, in the unlikely event that oil or linters from MON 88701 cotton would contain protein, PAT (bar) would not be consumed as a catalytically active protein in food.
To assess the potential for toxicity of PAT (bar), Monsanto conducted an acute oral toxicity study in which mice (ten male and ten female) were given a single dose of 1086 mg/kg bw of PAT (bar) or a comparable level of bovine serum albumin. Monsanto chose this level based on principles of hazard identification and margin of exposure. According to Monsanto, no treatment-related effects on survival, clinical observations, body weight gain, food consumption, or gross pathology were observed after 14 days in mice administered PAT (bar).
Based on the information discussed above, Monsanto concludes that PAT (bar) does not pose a significant health risk to humans or animals.
Food & Feed Uses of Cotton
Cotton (Gossypium hirsutum L.) is grown worldwide as a source of fiber for the textile industry. Cottonseed, which is a by-product of fiber production, is used in human food, animal feed, and a range of industrial products. Food uses of cottonseed include cottonseed oil and cotton linters. Cottonseed oil is highly refined to remove naturally occurring toxicants, gossypol, and cyclopropenoid fatty acids (CPFAs). Cottonseed oil is primarily consumed as a salad or cooking oil, for frying, in mayonnaise, and shortening. Cotton linters are short fibers that remain on cotton seeds after the long fibers have been removed at the ginning process for textile manufacturing. They are removed from the seeds and processed into pure cellulose, which is used in casings for bologna, sausages, and frankfurters and in ice cream and salad dressings.
Whole cottonseed, cottonseed meal, hulls, and cotton gin trash are used in animal feeds for cattle, sheep, goats, horses, poultry, swine, fish, and shrimp. Cottonseed meal is the product obtained after removal of oil from whole cottonseed flakes or cake and is used as a protein supplement in animal feed. Cottonseed hulls are used as a source of fiber in feeds.
Scope of Analysis
Monsanto assessed the similarities and differences in the compositions of acid de-linted cottonseed from MON 88701 cotton and the conventional control variety Coker 130 (hereafter referred to as the control). The compositional analyses included key nutrients and anti-nutrients. Monsanto also assessed the composition of acid de-linted cottonseed from nine reference cotton varieties (hereafter referred to as reference varieties). The reference varieties were different sets of four conventional cotton varieties per site grown under the same field conditions as MON 88701 cotton and control. Monsanto used data obtained from the nine reference varieties to generate a 99% tolerance interval for each component (hereafter referred to as the 99% tolerance interval). Monsanto used the data from the reference varieties to establish a range of natural variability for each component in conventional cotton varieties with a history of safe consumption grown under the same conditions.
Monsanto states that cottonseed was obtained from plants grown in four replicate plots, planted at each of eight field sites across the U.S. in a randomized complete block design during the 2010 growing season. MON 88701 plots were treated at the three-to five leaf stage with glufosinate herbicide and at the six-to-ten leaf stage with dicamba herbicide. Monsanto assessed the data sets using a mixed model of variance. Nine sets of statistical analyses were conducted: eight based on compositional data from each of the replicated field sites and one based on data combined across all eight field sites (combined site). The levels calculated from analytical data for each component obtained from individual sites and the data aggregated from all sites for MON 88701 cotton were compared with those of the control. Statistical significance was declared at 5% level (p ≤ 0.05). Monsanto excluded components from statistical analysis if fewer than 50% of the observed values were at or below the limit of quantitation. When a statistically significant difference in a component was detected between MON 88701 cotton and control, Monsanto assessed whether the difference was biologically meaningful from a food and feed safety or nutritional perspective. This analysis included assessing reproducibility across individual sites, magnitude of differences, and comparing the mean value for each component in MON 88701 cotton with the respective 99% tolerance interval and its levels in the published literature, including the International Life Sciences Institute Crop Composition Database (ILSI-CCD, 2011).
Results of analyses:
Monsanto reports the results of compositional analysis for key components in cottonseed: proximates (ash, carbohydrates (by calculation), fat, moisture, crude fiber, and crude protein), acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary fiber (TDF), 18 amino acids, fatty acids (C8-C22), nine minerals, and vitamin E. For the combined-site analysis, Monsanto observed statistically significant differences for some proximates (ash, carbohydrates (by calculation), moisture, and total fat), fiber (ADF, NDF, and TDF), three amino acids (arginine, methionine, and proline), two fatty acids (14:0 myristic acid and 18:2 linoleic acid), five minerals (calcium, magnesium, manganese, potassium, and zinc) and vitamin E in MON 88701 when compared with the control. Monsanto notes that differences between the means of these components for MON 88701 cotton and control were small. Mean values for MON 88701 cotton components that were statistically different from the control in the combined-site analysis were within the 99% tolerance intervals, except for methionine values. All component values, including methionine, were within the ranges reported in the literature, including ILSI-CCD.
Monsanto analyzed cottonseed for five anti-nutrients (dihydrosterculic acid, malvalic acid, sterculic acid, free gossypol, and total gossypol). Monsanto reports statistically significant differences between MON 88701 cotton and the control for dihydrosterculic acid, free gossypol, and total gossypol. The differences between the MON 88701 cotton and control mean values for each of these anti-nutrients were small in magnitude and the mean value for MON 88701 cotton for each of these components fell within the established 99% tolerance interval and within the range of literature values.
Summary of Compositional Analyses
Monsanto states that the compositional assessment supports its conclusion that MON 88701 cotton is compositionally equivalent to conventional cotton varieties. Therefore, Monsanto concludes that the differences in the observed levels of components between MON 88701 cotton and other cotton varieties are not meaningful for food and feed safety or nutrition.
FDA evaluated Monsanto's submission to determine whether MON 88701 cotton raises any safety or regulatory issues with respect to the intended modifications 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 concludes that, with the exception of the intended modifications (tolerance to dicamba and glufosinate herbicides), MON 88701 cotton and foods and feeds derived from it are not materially different in composition, safety, or any other relevant parameter from other cotton 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 88701 cotton to be complete.
Shayla West-Barnette, Ph.D.
1Monsanto states that MON 88701 cotton will be combined with other herbicide-tolerant (including glyphosate) events using traditional breeding techniques.
2The dmo coding sequence, optimized for expression in cotton, encodes for a DMO protein similar to that produced by S. maltophilia.
3P. hybrida DnaK gene encodes heat shock protein 70 (Hsp70).
4The analytical techniques included N-terminal sequence analysis, mass determination of the tryptic peptides by MALDI-TOF MS, SDS-PAGE, Western hybridization analysis, MON 88701 DMO activity analysis, and glycosylation analysis.
5MON 88701 cotton was treated at the 3-5 leaf stage with glufosinate herbicide at the label rate and at the 6-10 leaf stage with dicamba at the label rate.
6Due to a limited amount of tissue, moisture content was not measured for pollen; therefore, Monsanto reports pollen on a fresh weight basis only.
7The tissue samples used to measure PAT (bar) levels were the same as those used to measure DMO levels.