Biotechnology Consultation Note to the File BNF No. 000125
Return to inventory: Submissions on Bioengineered New Plant Varieties
Biotechnology Consultation - Note to File
Biotechnology Notification File No. 000125
Date: September 6, 2011
Subject: Dicamba Tolerant MON 87708 soybean
Keywords: Soybean, Glycine max, dicamba mono-oxygenase, MON 87708, Monsanto, herbicide tolerant, Stenotrophomonas maltophilia strain DI-6, OECD Unique Identifier MON-877Ø8-9
This document summarizes FDA’s evaluation of biotechnology notification file (BNF) No. 000125. In a submission dated November 9, 2010, Monsanto Company (Monsanto) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of the bioengineered MON 87708 soybean, which is tolerant to dicamba herbicide (dicamba). Monsanto provided additional information on February 25, 2011. FDA evaluated the information in Monsanto’s submissions to ensure that regulatory and safety issues regarding the food or feed derived from the new plant variety have been resolved prior to commercial distribution.
In our evaluation of BNF 000125, we considered all information provided by the notifier 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 87708 soybean is to produce dicamba-tolerant soybeans.1 Monsanto states that a conventional soybean variety was transformed with the dmo gene from Stenotrophomonas maltophilia strain DI-6. The dmo gene encodes precursor proteins that after processing form a multisubunit enzyme (dicamba mono-oxygenase, DMO) that rapidly demethylates dicamba (3,6-dichloro-2-methoxybenzoic acid) to the inactive metabolite 3,6-dichlorosalicylic acid (DCSA), thereby conferring dicamba tolerance to the soybean.
The purpose of this evaluation is to assess whether the developer has introduced a substance requiring premarket approval as a food additive or has unintentionally adulterated the food 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 herbicide, metabolic by-products, and residues in MON 87708 soybean that result from detoxification by the expression products of applied herbicides are considered pesticidal substances. Therefore, Monsanto has submitted a regulatory submission to EPA requesting the establishment of a tolerance for the use of dicamba on MON 87708 soybean.
Genetic Modification and Characterization
Monsanto transformed the recipient non-transgenic conventional soybean variety A3525 to produce MON 87708 soybean.
Transformation Vector and Method
Monsanto states that MON 87708 soybean was developed through Agrobacterium tumefaciens-mediated transformation of soybean A3525 meristem tissue utilizing transformation plasmid vector PV-GMHT4355. PV-GMHT4355 contains single copies of the T-DNA I and T-DNA II cassettes. The T-DNA I cassette (T-DNA I) contains the dmo coding sequence whose expression is regulated by the peanut chlorotic streak caulimovirus promoter, Tobacco Etch virus leader, pea Rubisco targeting sequence (small subunit), and the pea E9 3' non-translated region. The T-DNA II cassette contains the cp4 epsps coding sequence under the control of the figwort mosaic virus promoter, DnaK leader, CTP2 chloroplast targeting sequence, and the pea E9 3' non-translated region. Monsanto states that both T-DNA cassettes are delineated by left and right T-DNA border regions, which facilitate transformation.
Monsanto used self-pollination and segregation to isolate a soybean plant containing T-DNA I, but without the T-DNA II cassette; thus, MON 87708 soybean is dicamba-tolerant, but is susceptible to glyphosate.
Characterization, Inheritance, and Stability of the Introduced DNA
To characterize the introduced DNA, Monsanto conducted Southern hybridization analyses and DNA sequencing comparisons. Monsanto states that the results of the Southern hybridization analyses show that MON 87708 soybean contains a single copy of T-DNA I inserted at a single locus in the genome. Monsanto notes that Southern hybridization analyses also show that MON 87708 soybean lacks any detectable T-DNA II sequences or plasmid backbone sequences.
Monsanto states that the results of DNA sequencing analyses complement the Southern hybridization analyses and show that MON 87708 soybean contains a single copy of T-DNA I at a single insertion site. Monsanto states that the results of these analyses also show that each genetic element in the T-DNA I cassette is intact.
During the development of MON 87708 soybean, Monsanto recorded segregation data to assess the heritability and stability of the inserted coding sequence in the genome. The results of chi-square analysis of the segregation data from three generations show that T-DNA I is inherited according to Mendelian principles over multiple generations. The results of these analyses also confirm that MON 87708 soybean contains a single, intact copy of T-DNA I that is inserted into the soybean genome at a single locus.
Monsanto states that the same pattern was observed using Southern hybridization analyses of genomic DNA isolated from plants from five generations, further confirming that T-DNA I is stably integrated into the genome of MON 87708 soybean.
Monsanto performed open reading frame (ORF) bioinformatic analyses of the MON 87708 soybean 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 highly unlikely occurrence of translation of any of these ORFs, the polypeptide products would not be sufficiently similar with other proteins to indicate that they are potentially allergenic or toxic, nor would they constitute safety concerns.
Identity, Function, and Characterization
DMO is described as a Rieske non-heme iron oxygenase that functions by transporting electrons from ferredoxin to the non-heme iron domain. Monsanto states that diverse groups of organisms ranging from bacteria to plants produce oxygenases, and these enzymes are consumed by humans and animals. Monsanto states that the amino acid sequence of DMO in MON 87708 soybean (MON 87708 DMO) is similar to the amino acid sequences of bacterial mono-oxygenases.
Monsanto states that T-DNA I produces a single MON 87708 DMO precursor protein, which is post-translationally processed into two forms of the MON 87708 DMO protein and is targeted to the soybean chloroplasts using sequences from pea. Monsanto designates the forms of the protein as MON 87708 DMO protein and MON 87708 DMO + 27 protein (reflecting that this protein contains an additional 27 amino acids). Monsanto states that the active enzyme is a trimer that can be comprised of MON 87708 DMO protein, MON 87708 DMO + 27 protein, or a combination of both. In the notice, Monsanto states that the designation MON 87708 DMO refers to both proteins and all forms of the trimer, except for the characterization of the individual proteins using N-terminal sequence analysis, matrix-laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western hybridization analysis, protein activity analysis, and glycosylation analysis. Monsanto notes that MON 87708 DMO protein and MON 87708 DMO + 27 protein retain the sequence of DMO protein isolated from S. maltophilia except for an alanine at position 2 added for cloning purposes and a replacement of tryptophan with cysteine at position 112. Monsanto states that these changes would have minimal functional impact because they are sterically distant from the catalytic site.
Monsanto assessed whether there is potential for MON 87708 DMO protein to metabolize endogenous substrates present in MON 87708 soybean. Monsanto conducted in vitro experiments using a purified N-terminal histidine tagged DMO protein that, except for the histidine tag, is identical to DMO protein isolated from S. maltophilia. Monsanto tested the potential metabolic activity of the tagged DMO protein on a set of endogenous substrates (o-ansic, vanillic, syringic, ferulic, and sinapic acids) that were selected based on their structural similarity to dicamba and their presence in soybeans. Monsanto states that none of the tested substrates was metabolized by the tagged DMO protein used in these experiments. Thus, Monsanto states that these results demonstrate the specificity of MON 87708 DMO protein for dicamba as a substrate.
Monsanto also assessed whether MON 87708 DMO protein has the same specificity as the tagged DMO protein used in the in vitro experiments. Monsanto isolated MON 87708 DMO protein from MON 87708 soybean seed and tested the metabolic activity of the MON 87708 DMO protein with o-ansic acid (the endogenous compound with the greatest structural similarity to dicamba). Monsanto states that o-ansic acid was not metabolized by MON 87708 DMO and concludes that this result demonstrates that DMO, including MON 87708 DMO, is specific for dicamba.
Monsanto states that sufficient MON 87708 DMO was purified directly from MON 87708 soybean for the safety assessment, and therefore equivalence evaluation between soybean-produced and S. maltophilia-produced DMO was unnecessary. The physicochemical characteristics and functional activity of MON 87708 DMO were determined using various analytical techniques.2 The results of these analyses confirmed the identity of MON 87708 DMO, determined the apparent molecular weight and purity of the protein, demonstrated that the protein is functionally active, and showed that the protein is not glycosylated.
MON 87708 DMO levels in MON 87708 soybean were measured in replicate samples of root, leaf, forage, and seeds from five field sites using an enzyme-linked immunosorbent assay (ELISA). Monsanto reports that the range of MON 87708 DMO in MON 87708 soybean leaf, root, forage, and seeds was from 3.9 to 180 micrograms per gram dry weight (μg/g DW). The mean DMO protein concentrations (μg/g DW) in MON 87708 soybean were 17 to 69 for leaf at four vegetative stages, 6.1 for root, 53 for forage, and 47 for seeds.
Safety of the Donor Organism
Monsanto states that the dmo gene introduced into the parental soybean variety is derived from S. maltophilia. S. maltophilia is an aerobic, Gram-negative, environmentally ubiquitous bacterium, which is commonly found in aquatic environments, household environments, soil, and plants. Monsanto states that exposure to S. maltophilia is incidental due to its presence in foods such as ready-to-eat salads, vegetables, frozen fish, milk, and poultry. Published reports show that S. maltophilia can be found in healthy individuals without harmful effects. According to the published literature, S. maltophilia strains are known to 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 infections with S. maltophilia are rare and occur almost exclusively in hospital settings. Monsanto states that S. maltophilia has not been reported to be a source of allergens. Given the information discussed above, Monsanto states that S. maltophilia is a safe donor organism.
Assessment of Potential for Allergenicity and Toxicity
Monsanto performed studies to assess the potential for allergenicity and toxicity of MON 87708 DMO.
Monsanto notes that soybean is one of the eight major food allergens as defined in the Food Allergen Labeling and Consumer Protection Act of 2004. To assess the potential allergenicity of the MON 87708 DMO + 27 protein, Monsanto performed an amino acid sequence similarity search against the AD_2010 database (which contains amino acid sequences of allergens, gliadins, and glutenins) using the FASTA algorithm. Monsanto states that the results of amino acid sequence comparisons identified no relevant alignments (i.e., alignments that display an E-score of less than or equal to 1 x 10-5) between the MON 87708 DMO + 27 amino acid sequence and sequences in the AD_2010 database.3 Monsanto further states that no alignment met or exceeded the threshold of 35% identity over 80 amino acids.4 Monsanto notes that the MON 87708 DMO amino acid sequence is fully contained in the MON 87708 DMO + 27 amino acid sequence.
Monsanto also performed an eight amino acid sliding window search to specifically detect short linear polypeptide matches to known or suspected allergens in the AD_2010 database.5 Monsanto reports that no sequence alignments of eight contiguous amino acids were detected when the MON 87708 DMO + 27 amino acid sequence was compared to sequences in the AD_2010 database.
Monsanto states that the results of the bioinformatic analyses demonstrate the lack of both structurally and immunologically relevant similarities between the amino acid sequences of MON 87708 DMO and known allergens, gliadins, and glutenins. In addition, Monsanto states that all forms of MON 87708 DMO are rapidly degraded in vitro in simulated gastric fluid and in simulated intestinal fluid. Monsanto thus concludes that it is unlikely that the MON 87708 DMO will pose human health concerns related to food allergy or gluten sensitivity.
Monsanto performed an amino acid sequence similarity search on the MON 87708 DMO + 27 amino acid sequence against the TOX_2010 database using the FASTA algorithm. The TOX_2010 database contains amino acid sequences of proteins that may be harmful to human and animal health. Monsanto states that the results of the sequence comparisons found no relevant alignments (i.e., alignments that display an E-score of less than or equal to 1 x 10-5) between MON 87708 DMO + 27 protein sequence and sequences in the TOX_2010 database. Monsanto thus concludes that no sequence similarity exists between MON 87708 DMO and any known toxins or other biologically active proteins that would be harmful to human or animal health.
Monsanto conducted an acute oral toxicity study in mice. A single dose of 140 milligrams per kilogram body weight (mg/kg bw) of MON 87708 DMO was administered by gavage to five male and five female mice. Monsanto states that no treatment-related effects on survival, clinical observations, body weight, body weight gain, food consumption, or gross pathology were observed. Monsanto thus concludes that the No Observable Adverse Effect Level for MON 87708 DMO is 140 mg/kg bw.
Based on the information discussed above, Monsanto concludes that MON 87708 DMO poses no meaningful risks to human and animal health upon consumption.
Food & Feed Uses of Soybean
Monsanto describes historical and current uses of soybean in food and animal feed, and states that it intends to market MON 87708 soybean for the same commercial purposes as current transgenic and non-transgenic soybean varieties. Soybean seeds are processed primarily into oil and meal.
Soybean oil constitutes nearly 70% of consumption of edible fats and oils in the United States (U.S.), and is the second largest source of vegetable oil worldwide. Soybean products are also used as human foods and sources of food ingredients.
Soybean meal is the predominant use of soybeans in animal feed and is the most common supplemental protein source in U.S. livestock and poultry rations due to its nutrient composition, availability, and price. Soybean meal is processed in moist heat to inactivate trypsin inhibitors and lectins, which are antinutrients occurring in raw soybeans. Soybean forage is occasionally used in animal feed.
Thus, the food and animal feed uses of soybean and its processed products remain the predominant uses of soybeans.
Scope of Analysis
Monsanto analyzed the composition of forage and seed from MON 87708 soybean and A3525 soybean (hereafter referred to as the control) to assess similarities and differences. The compositional analysis included key nutrients and anti-nutrients. Monsanto also assessed the composition of forage and seed from twenty commercial reference soybean varieties (hereafter referred to as reference varieties), which were different sets of four conventional soybean varieties per site grown under the same field conditions as MON 87708 soybean and control. Monsanto used data derived from these 20 reference varieties to generate a 99% tolerance interval for each component (hereafter referred to as the 99% tolerance interval).6 Monsanto states that the data from the reference varieties reflect the natural variability in commercially grown soybean varieties.
Monsanto states that forage and seed were obtained from soybeans grown in three replicate plots, planted in a randomized complete block design, at each of five field sites across the U.S. during the 2008 growing season. Monsanto assessed the data sets using a mixed model of variance. Six sets of statistical analyses were conducted, five based on the data from each of the replicated field sites (individual-site), and the sixth based on data from all five field sites combined (combined-site). The levels calculated from analytical data for each component obtained from individual sites and the data aggregated from all sites for MON 87708 soybean were compared to that of the control. Statistical significance was declared at 5% level (p≤0.05). When a statistically significant difference in a component was detected between MON 87708 soybean and control, an analysis was conducted to assess whether the difference was biologically meaningful7 from a food and feed safety or nutritional perspective. This analysis included reproducibility across individual sites, magnitude of differences, and comparisons of MON 87708 soybean mean component levels to the 99% tolerance interval and levels in the published literature, including the International Life Sciences Institute Crop Composition Database (ILSI-CCD).8
Results of analyses:
Compositional analysis of soybean forage
Monsanto reports the results of compositional analysis for the key components in soybean forage (moisture, crude protein, crude fat, ash, carbohydrates by calculation, acid detergent fiber (ADF), and neutral detergent fiber (NDF)). A statistically significant difference between MON 87708 soybean and control (p<0.02) was observed for ADF in the combined site analysis. Monsanto reports that the mean level for MON 87708 soybean fell within the 99% tolerance interval. Monsanto also reports that this was within the combined literature range of levels for this component. Monsanto concludes that these differences are not biologically meaningful.
Compositional analysis of soybean seed
Monsanto reports the results of compositional analyses of 57 components in seed. Monsanto analyzed seed for proximates (moisture, crude protein, crude fat, ash, carbohydrates by difference), ADF, NDF, crude fiber, fatty acids (C8-C22), 18 amino acids, vitamin E, isoflavones (daidzein, genistein, and glycitein), and anti-nutrients including phytic acid, trypsin inhibitors, lectin, raffinose, and stachyose. Of the measured components in MON 87708 soybean seed, 14 fatty acids had more than 50% of the observations below the assay limit of quantitation. These components were not included in the statistical analyses. For the combined-site analyses, no statistically significant differences were observed between MON 87708 soybean and control in the mean levels of moisture, crude fat, 6 amino acids (alanine, lysine, methionine, serine, threonine, and tryptophan), and 3 fatty acids (18:0 stearic acid, 20:0 arachidic acid, and 20:1 eicosenoic acid). Statistically significant increases were observed in the mean levels for ash, carbohydrates by difference, crude fiber, ADF, and NDF, and there was a statistically significant decrease in mean levels for crude protein. Monsanto reports that all of these levels for MON 87708 soybean fell within the 99% tolerance intervals and within the combined range of literature levels. Statistically significant differences between MON 87708 soybean and control were found for 12 amino acids (arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, tyrosine, and valine). Monsanto reports that the mean levels for these amino acids were within the 99% tolerance intervals and within the range of combined literature levels. Statistically significant differences between MON 87708 soybean and control were also observed (p<0.001) for five fatty acids (palmitic, oleic, linoleic, linolenic, and behenic acids). Monsanto reports that the mean levels for these fatty acids for MON 87708 soybean were within the 99% tolerance interval and within the combined range of literature levels. A statistically significant difference between MON 87708 soybean and control was measured for vitamin E. Monsanto reports that the mean levels for vitamin E for MON 87708 soybean were within the 99% tolerance interval and within the range of literature levels. In addition, a small, but statistically significant, increase in daidzein was observed in MON 87708 soybean when compared to the control but the values were within the 99% tolerance interval and within the range of literature values. Monsanto concludes that these differences are not biologically meaningful.
Small, but statistically significant, decreases in phytic acid, raffinose, and stachyose were observed in MON 87708 soybean when compared to the control. Monsanto reports that the mean levels for these anti-nutrients in MON 87708 soybean fell within the 99% tolerance intervals and within the range of literature levels. Thus, Monsanto concludes that the small differences are not biologically meaningful.
Monsanto states that the compositional assessment supports the conclusion that MON 87708 soybean is compositionally equivalent to conventional soybean varieties. Monsanto concludes that the small differences in the levels of the components listed above are not considered biologically meaningful for food and feed safety or meaningful from a nutritional perspective.
Monsanto conducted a study to determine whether the transformation process may have increased the overall allergenicity of MON 87708 soybean compared to conventional soybean. Using sera from clinically documented, soybean-allergic patients, Monsanto conducted immunoglobulin E (IgE) ELISA using protein extracts from MON 87708 soybean and control. Monsanto reports that soybean-specific IgE binding to endogenous allergens in MON 87708 soybean and control are comparable with IgE binding to conventional soybeans currently on the market. Therefore, Monsanto concludes that MON 87708 soybean does not increase exposure to endogenous allergens from that of conventional soybeans.
Monsanto has concluded that, with the exception of the intended modification (dicamba tolerance), MON 87708 soybean and the foods and feeds derived from it are not materially different in composition, safety, or any other relevant parameter from other soybean 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 87708 soybean to be complete.
1Monsanto states that MON 87708 soybean will be combined with the glyphosate-tolerant MON 89788 soybean (Roundup Ready 2 Yield® soybean) to enable a weed management program to control a broad spectrum of weed species.
2The analytical techniques discussed in the submission include N-terminal sequence analysis, mass determination of the tryptic peptides by MALDI-TOF MS, SDS-PAGE, Western hybridization analysis, MON 87708 DMO protein activity analysis, and glycosylation analysis.
3The E-score was set at <1 x 10-5 to ensure that sequences have sufficient sequence similarity to infer homology.
4These criteria can be found in the guidelines for the evaluation of the potential allergenicity of introduced proteins, published in 2003 by the Codex Alimentarius Commission.
5An amino acid sequence may be considered to have allergenic potential if it has an exact sequence identity of at least eight contiguous amino acids with a potential allergen epitope (Hileman et al., 2002; Metcalfe et al., 1996).
6A 99% tolerance interval represents, with 95% confidence, 99% of the levels contained in the population of commercial conventional soybean varieties.
7FDA considers biologically meaningful to connote that there is a notable impact on dietary needs or requirements, or an impact that otherwise would affect health.
8Monsanto used version 3.0 (accessed on 8-27-06) of the ILSI-CCD in its analysis. The database is maintained by ILSI and can be accessed at http://www.cropcomposition.org/.