Biotechnology Consultation Note to the File BNF No. 000117
Return to inventory: Completed Consultations on Foods from Genetically Engineered Plant Varieties
Biotechnology Consultation - Note to File
Biotechnology Notification File No. 000117
July 26, 2012
MON 87769 Stearidonic acid (stearidonate) soybean
Soybean; Glycine max; altered composition; stearidonic acid (SDA); stearidonate; omega-3 fatty acids; desaturases; Primula juliae; primrose; Pj.D6D; Pj delta(Δ)6D desaturase; Neurospora crassa; Nc.Fad3; NcΔ15D desaturase; Agrobacterium sp. strain CP4; cp4 epsps; 5-enolpyruvyl shikimate-3-phosphate synthase; CP4 EPSPS; MON 87769; Monsanto Company; OECD Unique Identifier MON-87769-7
This document summarizes our evaluation of biotechnology notification file (BNF) No. 0117. In a submission dated March 20, 2009, Monsanto Company (Monsanto) submitted a safety and nutritional assessment of a bioengineered soybean with an altered oil composition transformation event MON 87769 (hereafter referred to as MON 87769 soybean). Monsanto provided additional information on October 15, 2009; November 8, 2010; February 10, 2011; March 16, 2011; and May 4, 2012. FDA evaluated the information in Monsanto's submission to ensure that regulatory and safety issues regarding the food and feed derived from the new plant variety have been resolved prior to commercial distribution.
In our evaluation, 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 technical effect of the modification in MON 87769 soybean is to produce soybean oil with an altered fatty acid profile. Specifically, MON 87769 soybean produces stearidonic acid or stearidonate (SDA), an omega-3 fatty acid that is not normally synthesized by soybeans, at approximately 20-30% of total fatty acids. To accomplish this objective Monsanto introduced two genes encoding desaturase enzymes: the delta (Δ)6 desaturase gene (Pj.D6D) from Primula juliae (primrose) and Δ15 desaturase gene (Nc.Fad3) from the mold Neurospora crassa that encode the PjΔ 6D and NcΔ 15D proteins, respectively. Desaturases introduce double bonds into fatty acids at specified positions. Δ6 desaturase preferentially converts alpha-linolenic acid (ALA) to SDA. However, dependent on linoleic acid (LA) levels, Δ6 desaturase may also convert LA to gamma-linolenic acid (GLA), an omega-6 fatty acid. Δ15 desaturase, expressed in conjunction with Δ6 desaturase, increases the ALA substrate pool for generating SDA production in MON 87769 soybean and lowers the LA substrate pool for GLA production. Monsanto states that the oil derived from MON 87769 soybean is intended to serve as an alternative sustainable source of omega-3 fatty acids.
The purpose of this evaluation is to assess whether the developer has introduced into food a substance requiring premarket approval as a food additive or has raised other issues under the Federal Food, Drug, and Cosmetic Act (FD&C Act).
Genetic Modification and Characterization
Parental Variety and Transformation Method
Monsanto used Agrobacterium-mediated transformation of soybean meristem tissue derived from A3525 soybean, a non-transgenic elite commercial variety.
Monsanto describes the plasmid vector PV-GMPQ1972 as containing two transfer DNA (T-DNA) regions each delineated by a right and left border region. The first T-DNA region (T-DNA I) contains two expression cassettes: the Pj.D6D gene expression cassette and the Nc.Fad3 gene expression cassette. The Pj.D6D gene expression cassette encodes a single polypeptide designated PjΔ6D. The Pj.D6D coding sequence is under the control of the seed-specific promoter and leader sequence P-7Sα′ from the soybean Sphas1 gene.1 Following the coding sequence is the 3′ untranslated region of the tml gene from the Agrobacterium tumefaciens octopine-type Ti plasmid. The Nc.Fad3 gene expression cassette encodes a single polypeptide, designated NcΔ15D. The Nc.Fad3 coding sequence is under the control of the seed-specific promoter and leader sequence P-7Sα from the soybean Sphas2 gene.2 Following the coding sequence is the 3′ untranslated region of the pea (Pisum sativum) ribulose-1,5-bisphosphate carboxylase small subunit (rbcS2) gene (designated T-E9).
Monsanto states that the second T-DNA (T-DNA II) region contains the cp4 epsps gene expression cassette that encodes the CP4 EPSPS protein (5-enolpyruvyl shikimate-3-phosphate synthase protein from Agrobacterium sp. strain CP4) with appropriate regulatory sequences. This provides tolerance to the action of the herbicide glyphosate, thereby acting as a selectable marker for transformation.
The use of the two T-DNAs allows for the insertion of the T-DNAs into two independent loci within the genome of the plant. Following selection of the transformants that contain both T-DNAs, T-DNA II was removed by conventional breeding. Therefore, the selectable marker (cp4 epsps) is not present in MON 87769 soybean.
Characterization, Inheritance, and Stability of the Introduced DNA
Monsanto characterized the insert in MON 87769 soybean using restriction enzyme digestion of genomic DNA followed by Southern blot analyses. Monsanto reports that MON 87769 soybean has a single insert of T-DNA I containing the Pj.D6D and Nc.Fad3 gene expression cassettes and that the nucleotide sequence and the organization of the insert are maintained as in the introduced T-DNA. Monsanto confirmed the nucleotide sequence and organization of the inserted DNA by DNA sequencing analyses. Monsanto confirmed the absence of plasmid backbone and T-DNA II expression cassette by Southern blot analysis.
Monsanto performed bioinformatic analyses of the MON 87769 soybean insert and flanking genomic DNA sequences to determine whether insertion of the introduced DNA created potential open reading frames (ORFs) that may encode for a potential toxin, allergen, or biologically active polypeptide. The same analyses were performed for alternate reading frames within the coding sequences of the expression cassettes. The analyses provided no evidence to support that the sequences spanning the genomic-insert DNA junctions or alternate reading frames of the coding sequences of the expression cassettes are translated to yield polypeptide products. Based on bioinformatic analyses, Monsanto concludes that even in the unlikely occurrence of translation of any ORFs, the resulting putative polypeptides would not share homology with known allergens, toxins, or biologically active polypeptides and would not constitute a safety concern.
Using Southern blot analysis with overlapping probes that span the entire T-DNA I region to examine the structure and stability of the insert, , the stability of the insert was characterized across four generations. Monsanto confirmed that a single copy of the insert in MON 87769 soybean is stably maintained across multiple generations.3 Transgenerational segregation of T-DNA I in MON 87769 soybean was confirmed by performing a Chi-square analysis of zygosity data collected for three generations. Monsanto reports that the inheritance of insert follows the expected Mendelian pattern of segregation for a single locus.
Identity and Function of Introduced Proteins
PjΔ6D and NcΔ15D are fatty acid desaturases. Fatty acid desaturases are non-heme iron-binding enzymes that convert a single bond between two specific carbon atoms in a fatty acyl chain into a double bond. Fatty acid desaturases act at a defined carbon atom of fatty acyl chains with strict regioselectivity and stereoselectivity. The PjΔ6D and NcΔ15D proteins are members of a family of integral membrane fatty acid desaturases found in all eukaryotic organisms. PjΔ6D and NcΔ15D are localized to the membranes of the endoplasmic reticulum and require a close association with electron transport proteins to function.
The PjΔ 6Ddesaturase protein expressed in MON 87769 soybean is identical to the native protein produced in P. juliae (primrose). PjΔ 6D contains 446 amino acids and has an apparent molecular weight (m.w.) of 46 kiloDaltons (kDa).
The NcΔ 15D protein is identical to the native protein produced in N. crassa except for a single amino acid replacement of threonine to alanine at the first amino acid after the start codon. This change was introduced to facilitate the insertion of the gene into the plant transformation vector. NcΔ15D contains 429 amino acids and has an apparent m.w. of 46 kDa.
Purification and Characterization of Introduced Proteins
Monsanto reports that studies were conducted on PjΔ 6D and NcΔ 15D desaturases isolated directly from MON 87769 soybean. Monsanto states that the purification of integral membrane proteins is technically challenging and requires removal of membranes and replacement of lipids surrounding the hydrophobic, membrane-spanning regions of a protein with an appropriate detergent to keep the protein in solution.
Monsanto used immature seeds from MON 87769 soybean as the source for purification of the PjΔ 6D and NcΔ 15D desaturases. A panel of detergents was tested for the ability to release these proteins from MON 87769 soybean seed membranes while maintaining the solubility of the proteins. Monsanto states that Fos-choline 124 was used in the purification of the PjΔ6D and NcΔ15D desaturases. After the proteins were solubilized from the membranes, multiple chromatographic steps were applied, followed by immuno-affinity purification to further purify the proteins. Monsanto states that standard precautions were taken to retain the integrity of the proteins during purification.5
To characterize the purified PjΔ6D and NcΔ15D desaturases, Monsanto performed a number of analyses to confirm the identity of the proteins.6 Monsanto reports that the analyses confirm the identity of the PjΔ6D and NcΔ15D proteins isolated from MON 87769 soybean.
Monsanto states that confirmation of the functions of the PjΔ6D and NcΔ15D proteins was provided by several lines of evidence: MON 87769 soybean accumulates SDA as well as GLA, which are novel in the context of soybean lipid biosynthesis; functional activity of each protein was observed in a yeast expression system; and, in an in vitro assay for PjΔ6D, SDA was produced from a precursor using a crude extract of immature MON 87769 soybean seed.7
Antibodies to PjΔ6D and NcΔ15D Proteins
Monsanto describes that antibodies to PjΔ6D and NcΔ15D proteins, produced in goats, were raised against peptide fragments of the two proteins. For the PjΔ6D protein, Monsanto used a 12 amino acid residue peptide fragment located near the C-terminus to elicit antibody production. For the NcΔ15D protein, Monsanto isolated a 19 amino acid residue fragment from the central region of the NcΔ15D protein to elicit antibody production. Antibodies specific to the full length proteins were developed and used for digestibility studies.
Protein Expression Level
Levels of PjΔ6D and NcΔ15D proteins in MON 87769 soybean were determined. Both proteins are membrane-bound proteins, which require detergent extraction. Detergents interfere with antibody interactions; therefore, quantitation using standard ELISA techniques is not practicable. Consequently, Monsanto used SDS-PAGE, which separates the protein from the detergent, followed by Western blot analysis using an antibody specific to a fragment of the soluble portion of the protein. Densitometric analysis of PjΔ6D protein- and NcΔ15D protein-specific immunoblots yielded quantifiable data based on the use of purified protein standards. Limits of quantitation for the two proteins in each plant tissue type were determined as the lowest amounts of the standards that could be reliably determined, and limits of detection were defined as the lowest amounts of these proteins that could be observed visually using labeled probes. Tissue samples were collected from five field locations in the United States (U.S.) during the 2006 soybean growing season. At each site, three replicated plots of MON 87769 soybean were grown using a randomized complete block field design. Over-season leaf (at various stages of development), forage, root, immature seed, and mature seed were collected from each replicated plot in all field sites. Protein concentrations were calculated on a microgram (µg) per gram (g) fresh weight (FW) basis. (After determining moisture content, Monsanto calculated the proteins' concentrations on a dry weight (DW) basis as well.)
As the expression of both proteins is controlled by seed-specific promoters, neither protein was detected in over-season leaf and root tissues, but, as expected, both proteins were detected in immature seed and mature seed. Both proteins were also detected in forage, which Monsanto attributes to small amounts of immature seed in forage. Expression of the desaturase proteins is higher during seed development; therefore, the levels of PjΔ6D and NcΔ15D proteins are higher in immature seeds. The mean concentrations of PjΔ6D protein in immature seed, mature harvested seed, and forage were 27, 1.7 and 4.3 µg/g FW (100, 1.8, and 16 µg/g DW), respectively. The mean concentrations of NcΔ15D protein in immature seed, mature harvested seed, and forage were 55, 9.5 and 3.7 µg/g FW (200, 10, and 14 µg/g DW), respectively. As expected, neither protein was detected in control soybean.
Monsanto provides estimates of dietary exposure to the PjΔ6D and NcΔ15D proteins from consumption of MON 87769 soybean products by humans and animals. Monsanto assumes that most exposure to the proteins will come from consumption of soybean meal.
Human Exposure: Monsanto used the Dietary Exposure Evaluation Model (DEEM-FCID version 2.03, Exponent Inc.) and food consumption data from the 1994-1996 and 1998 U. S. Department of Agriculture Continuing Survey of Food Intakes by Individuals (CSFII) to estimate exposure. Monsanto considers that soybean oil would contain little protein and thus would not be a significant source of these proteins. Monsanto considers it likely that the soybean meal from MON 87769 soybean would be combined with other soybean meal in processed products; however, for a conservative estimate of dietary exposure, they have assumed that all soybean meal would be from MON 87769 soybean. Monsanto based its calculations on the levels in mature seed (discussed above), correcting the levels for the amount of soybean meal obtained from seed. Monsanto estimates the 95th percentile acute intake for the general population of PjΔ6D and NcΔ15D proteins at 0.00016 milligrams per kilogram body weight per day (mg/kg bw/d) and 0.0008 mg/kg bw/d, respectively. Monsanto estimates acute intake for non-nursing infants of PjΔ6D and NcΔ15D proteins at 0.006 mg/kg bw/d and 0.036 mg/kg bw/d, respectively.
Animal Exposure: Monsanto calculated exposure of the PjΔ6D and NcΔ15D proteins in animals. Monsanto notes that the yield of soybean meal from seeds is 74%. Monsanto will limit the use in animal feed of MON 87769 soybean to soybean meal. Monsanto considers that the highest consumption level of soybean meal would be in broiler chickens, as the typical broiler chicken diet contains 18 g protein/kg bw/d. Monsanto calculates the highest level of dietary intake in broilers of PjΔ6D and NcΔ15D proteins as 0.117 mg/kg bw/day and 0.914 mg/kg bw/day, respectively, and considers that the dietary intake by other animals would be lower, because other animals consume less protein per kilogram of bodyweight.
Potential for Toxicity of the Introduced Proteins
Monsanto states that it has considered the potential for toxicity of the PjΔ6D and NcΔ15D proteins8 and considers that proteins homologous to the PjΔ6D and NcΔ15D proteins are present in all kingdoms of organisms, concluding that such proteins are familiar to the diets of humans and animals. Monsanto states that these proteins lack structural similarity to known toxins or biologically active proteins known to have adverse effects on human or animal health. Monsanto also notes that the expression levels of the two proteins are low and consumption is likely to be negligible. Both PjΔ6D and NcΔ15D proteins were readily digested in simulated gastric and simulated intestinal fluids.
Monsanto reports that when mice were administered acute doses of 4.7 mg/kg bw of PjΔ6D protein and 37.3 mg/kg bw of NcΔ15D protein, no adverse health effects were observed. Monsanto notes that based on these results. it calculated margins of exposure (MOE) for acute dietary intake of PjΔ6D of approximately 29,000 for the general population and 800 for non-nursing infants. The MOE for acute dietary intake of NcΔ15D were estimated to be 47,000 and 1,000 for the general population and non-nursing infants, respectively. Consequently, Monsanto concludes that no meaningful risks to human health would be likely from exposure to foods containing soybean meal from MON 87769 soybean.
Potential for Allergenicity of the Introduced Proteins
Monsanto discusses a weight of the evidence approach in assessing the potential for allergenicity of the introduced proteins. Monsanto first discusses that the source of PjΔ6D protein, P. juliae, while not generally consumed in food, is not a known source of food allergens. Monsanto reports that the source of the NcΔ15D protein, N. crassa, is a ubiquitous fungus considered to be non-pathogenic and non-allergenic. Monsanto also notes that the concentrations of these proteins in mature MON 87769 soybean seeds is very low (0.00043% DW and 0.00239% DW of total protein for the PjΔ6D and NcΔ15D proteins, respectively). Monsanto relates the bioinformatic comparison of the PjΔ6D and NcΔ15D proteins to known allergens (1) using a FASTA sequence alignment program to assess if 35 or more percent identity occurs over any sequence of 80 amino acid residues and (2) using the ALLERGENSEARCH program (release date 2008, assembled with data from the publicly available FARRP database AD8, release date January, 2008) to identify any matches of 8 contiguous amino acids, over a sliding window, to sequences present in known allergens. None of the proteins in the database met or exceeded the threshold of 35% identity over 80 amino acids with PjΔ6D or NcΔ15D proteins. Monsanto also relates that no alignments had E scores (incorporating percent identity and length of alignment) below one, concluding that no alignments were relevant from an allergenic assessment perspective. Although noting that the relevance of the 8 contiguous amino acid matches for assessing allergenic epitopes has not been validated, Monsanto reports that there were no identical 8 contiguous amino acid matches between PjΔ6D protein and known allergens. There was one 8 contiguous amino acid match between NcΔ15D protein and a protein from wheat, a known source of allergens. Monsanto identifies the match as a series of eight serine residues in a wheat carboxypeptidase protein. Monsanto notes that the polyserine sequence is not known to confer a unique structure and is also common to many other proteins from non-allergenic food sources that are commonly consumed and thus, the sequence is unlikely to be an indicator of allergenicity.
Monsanto also relates the results from an analysis of the digestibility in a simulated gastric fluid (SGF) assay containing pepsin. Monsanto estimates that over 99% of PjΔ6D was digested within 30 seconds of incubation with SGF, and no intact protein was detectable at any subsequent time points. Similarly, for NcΔ15D, 97% was estimated to be digested within 30 seconds of incubation with SGF. Monsanto also performed similar assays using simulated intestinal fluid and obtained similar results. Monsanto concludes that both proteins are readily digestible. Monsanto concludes that neither protein would pose a significant risk for eliciting an allergic response.
Food and Feed Use
Soybean (Glycine max) is grown around the world for a variety of food, feed, and industrial uses. Customarily, soybean seeds are processed into oil and meal. Typical soybean oil is rich in polyunsaturated fatty acids and is commonly used as a salad and cooking oil and in the production of margarine and other food ingredients. A small fraction of soybean meal is further processed into soy flours and soy proteins for a variety of food uses. Because of the altered oil composition of MON 87769 soybeans, it is unlikely that the beans would be used for traditional food uses like tofu, tempeh, and other products.
The preponderance of soybean meal is used in animal feed, primarily in poultry, swine, and beef and dairy cattle diets. Soybean meal is processed to remove the oil (defatted), and then treated in moist heat to inactivate trypsin inhibitors and lectins, which are anti-nutrients occurring in raw soybeans. Monsanto states that soybean meal from MON 87769 soybean will be used in animal feed, but that it currently does not intend for oil, whole soybeans, or forage from MON 87769 soybean to be included in animal feed. Monsanto notes that soybean forage is typically fed to cattle.
Monsanto states that oil extracted from MON 87769 soybean is intended to be a sustainable source of omega-3 fatty acids for human food. MON 87769 soybean has been modified to produce SDA. In humans and other animals, SDA is a metabolic precursor to the long chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Monsanto reports that the results of human and animal studies show that 1 gram of dietary SDA increases the tissue concentration of EPA to an equivalent extent as 200-300 mg of dietary EPA. The oil from MON 87769 soybeans contains approximately 20-30% SDA. Oil from MON 87769 soybean may be used as a replacement for omega-3 containing oils or omega-3 fatty acids in human food.9
Scope of Analyses
Monsanto states that compositional analyses were conducted on harvested seed and forage from MON 87769 soybean to assess whether the nutrient and anti-nutrient levels are comparable to those in conventional soybeans, except for the intended change in fatty acid composition of harvested seed. Harvested seed samples were also collected and prepared as soybean processed fractions: defatted toasted (DT) soybean meal; refined, bleached, and deodorized soybean oil (RBD oil); protein isolate; and crude lecithin fractions.
Monsanto analyzed the composition of seed and forage from MON 87769 soybean; from A3525 soybean, a conventional soybean that has background genetics representative of MON 87769 soybean (hereafter called control); and from 10 commercially available conventional soybean varieties (hereafter referred to as reference varieties).
Because Monsanto's intended change in MON 87769 soybean variety alters the fatty acid composition of the soybean, Monsanto expected that SDA, GLA, trans-SDA, and trans-ALA levels in MON 87769 soybean would be increased relative to the other soybean varieties.
Study Design - Compositional Analyses
Monsanto analyzed seed and forage derived from soybean varieties grown under replicated field conditions at five U.S. locations during the 2006 growing season. Levels of various components were assessed from MON 87769 soybean, the control, and the reference varieties. A range of observed values from the reference soybean varieties was determined for each analytical component and the data were used to calculate a 99% tolerance interval.10 Compositional analysis of harvested soybean included proximates (moisture, crude fat, crude protein, ash, and carbohydrates by calculation), acid detergent fiber (ADF), neutral detergent fiber (NDF), amino acids, fatty acids, isoflavones (daidzein, glycitein, and genistein), vitamin E, trypsin inhibitors, phytic acid, lectin, raffinose, and stachyose. Compositional analysis on soybean forage included proximates and fiber (ADF and NDF).
Monsanto performed six sets of statistical analyses; five were based on the data from each of the replicated field sites (individual-site), while the sixth analysis was based on data aggregated from all five field sites (combined-site). In all, 75 different analytical components, 68 in harvested seed and seven in forage were measured. Of the measured components, 26 in harvested seed had more than 50% of the observations below the assay limit of quantitation (LOQ) and thus were excluded from statistical analysis. Statistical analyses were conducted for 49 components (42 in harvested seed and seven in forage). The overall data set was examined for evidence of statistical significance using a mixed model analysis of variance. The compositional data set was examined for evidence of statistically significant differences between MON 87769 and control soybeans. Statistically significant differences were determined at the 5% level of significance (p<0.05). When a statistically significant difference in a component was detected between MON 87769 and control soybeans in the combined-site comparison, an analysis was conducted to assess whether the difference was biologically meaningful from a food and feed safety or nutritional perspective. This analysis included reproducibility across individual sites, magnitude of differences, and comparisons of MON 87769 soybean mean value with the 99% tolerance interval for the population of reference varieties (grown concurrently) and values in published literature and the International Life Sciences Institute Crop Composition Database (ILSI-CCD).11
Results of Analyses:
Compositional analyses of soybean seed
Fatty Acids – Intended Compositional Change
Monsanto reports that, as intended, SDA was produced in MON 87769: the levels of SDA in MON 87769 soybean seed ranged from 16.8% to 33.9% of total fatty acids with a mean of 26.1%. Further, as expected, GLA was produced in MON 87769 soybean seed: the levels of GLA in MON 87769 soybean seed ranged from 6.1 to 8.0% of total fatty acids with a mean of 7.1%. Monsanto reports low, but detectable, levels of two other fatty acids, trans-SDA (mean = 0.2%, range = 0.06 - 0.26%) and trans-ALA (mean = 0.4%, range = 0.38 - 0.48%). Also, as expected, SDA, GLA and the two trans-isomer components were not detected in the conventional soybean seed; therefore, no statistical comparisons for these components in MON 87769 and control soybeans were made.
Other Fatty Acids
The eight fatty acids common to both MON 87769 and control soybeans were analyzed as a percent of total fatty acids. Monsanto reports statistically significant differences for six fatty acids: oleic acid, LA, ALA, arachidic acid, palmitic acid, and behenic acid in MON 87769 soybean compared to the control. Monsanto states that these differences were relatively small in magnitude. The mean values and ranges for MON 87769 soybean seed were within the 99% tolerance interval, except for LA, which, as expected, was lower than in the control and reference varieties.
Protein and Amino Acids
Monsanto compared the concentrations of protein and individual amino acids in soybean seed from MON 87769 soybean with the control. There was a slight increase in crude protein concentration (<10%) in MON 87769 soybean as compared to the control. Monsanto compared the levels of individual amino acids between MON 87769 and control soybeans and reports statistically significant, but small, differences for 17 amino acids. The mean component values were all within the 99% tolerance interval for the population of reference varieties and also within the range of values found in the published literature and ISLI-CCD. Therefore, Monsanto considers that these differences were not biologically meaningful from a safety or nutritional perspective.
Proximates and Fiber
Monsanto reports that of the five proximate and two fiber components measured in seed, there were statistically significant differences in the levels of carbohydrates (by difference) and crude protein between MON 87769 and control soybeans. However, these differences were small in magnitude and the mean component values were all within the 99% tolerance interval for the population of reference varieties and also within the range of values found in the published literature and ISLI-CCD. Therefore, Monsanto considers that these differences were not biologically meaningful from a safety or nutritional perspective.
Monsanto reports that there is no significant difference in the level of vitamin E between MON 87769 and control soybeans.
Anti-nutrients and Isoflavones
Monsanto reports no statistical differences were observed between MON 87769 and the control soybean for the anti-nutritional factors analyzed (trypsin inhibitors, lectins, stachyose, raffinose, and phytic acid).
Monsanto reports that the mean and range values for the three isoflavones (i.e., genistein, daidzein, and glycitein), were lower in MON 87769 soybean seed compared to values in the control. However, the range of values for the three components were all within the 99% tolerance interval derived from the reference varieties. Therefore, Monsanto considers that these differences were not biologically meaningful from a safety or nutritional perspective.
Monsanto conducted a study to determine the binding levels of IgE antibody collected from clinically documented, soybean allergic patients to protein extracts prepared from MON 87769 soybean, the control, and 24 commercial soybean varieties that served to establish a range in IgE binding. Monsanto states that the results of this study demonstrated that the levels of endogenous allergens in MON 87769, and control soybeans are comparable to the levels of endogenous soybean allergens in the soybean varieties currently on the market.
Compositional analyses of soybean forage
Although Monsanto does not currently intend for forage from MON 87769 soybean to be used in animal feed, they provided information on the composition of MON 87769 soybean forage as part of their submission. Monsanto reports that no statistically significant differences were observed between MON 87769 and control soybeans for five proximate and two fiber components in soybean forage.
Compositional analyses of processed soybean products
DT Soybean Meal
Monsanto compared the composition of DT soybean meal from MON 87769 and control soybeans. Four amino acids (aspartic acid, glutamic acid, histidine, and tryptophan), ADF, and carbohydrates (by calculation) values were statistically different. The magnitude of the differences was small and the mean and the range of the values for these six components were all within the 99% tolerance interval for the reference varieties.
RBD oil samples from MON 87769 soybean, the control, and eight reference varieties were analyzed for fatty acids and vitamin E. As expected, SDA, GLA, and trans-SDA were found in RBD oil from MON 87769 soybean and were present at the same relative proportion to those observed in seed of MON 87769 soybean. Also as expected, SDA, GLA, and trans-SDA are not detected in RBD oil from control or reference varieties as they are not present in the soybeans.
Unlike in harvested seed, trans-ALA was observed in the RBD oil of the control and MON 87769 soybeans. The presence of trans-ALA in the control RBD oil is likely due to the effects of processing. As expected, the level for trans-ALA was higher for MON 87769 soybean (mean = 0.51% of total fatty acids) than for the control (mean = 0.14% of total fatty acids). Monsanto concludes that, as the levels in RBD oil from MON 87769 soybean are comparable to levels of trans fatty acids in commercial soybean oil, these levels would raise no safety concerns.
Monsanto notes that soybean protein isolate can be used in a wide range of food applications, including soup, sauce bases, energy bars, nutritional beverages, infant formula, and dairy replacements. Soybean protein isolate contains 90% protein. Monsanto analyzed protein isolate for amino acids and moisture. Monsanto reported a statistically significant, but small, difference in the concentration of leucine, which Monsanto does not consider to be biologically meaningful. The mean and range of values for amino acids and moisture obtained from MON 87769 soybean protein isolate were comparable to values from the control and also were within the 99% tolerance interval for the population of conventional reference varieties.
Crude lecithin prepared from MON 87769 soybean, control, and conventional soybean reference varieties were analyzed for four individual phosphatides, i.e., α-phosphatidic acid, α-phosphatidyl-choline, α-phosphatidyl-ethanolamine, and α-phosphatidylinositol. No statistically significant differences were observed in the phosphatide levels in crude lecithin obtained from MON 87769 and the control soybeans. All were within the 99% tolerance interval for the population of conventional reference varieties.
Fatty Acid Intake
Monsanto discusses the fatty acids from MON 87769 soybean-derived products and their impact on human and animal nutrition. As discussed above, SDA is an intermediate in the metabolism of ALA to long chain omega-3 fatty acids in mammals. SDA is found in various food sources, most notably fish oils. GLA is a metabolite in the conversion of LA to arachidonic acid in mammals. GLA is present in a variety of food sources and human breast milk. Monsanto states that published nutritional and toxicological studies on SDA and GLA in humans and animals support the safety of the intended uses of MON 87769 soybean and products derived therefrom.
Monsanto reports values for total trans fatty acid (TFAs) levels in MON 87769 soybean oil as approximately 0.6% of total fatty acids. Total TFAs in MON 87769 soybean oil are within the published range of total TFA content of commercial refined soybean oils. TFAs arise in non-hydrogenated vegetable oils during oil refining. Monsanto states that the contribution of MON 87769 soybean to the overall dietary TFA intake will be minimal relative to commonly experienced human dietary intakes. Thus, trans-SDA and trans-ALA do not raise safety concerns.
Although LA is an essential nutrient and the levels of LA in MON 87769 soybean and soybean oil are low, Monsanto notes that daily human LA intake exceeds the amount of LA needed to support human health. Also, the decrease in LA in MON 87769 soybean will not affect animal nutrition, because defatted soybean meal (the primary ingredient derived from soybean for animal feed) is not a significant contributor of fatty acids to animal diets. Consequently, Monsanto concludes that the decrease in LA in MON 87769 soybean will not have a negative impact on human or animal nutrition and health.
Animal Studies with DT Soybean Meal
Monsanto reports the results of a 42-day broiler chicken feeding study comparing birds fed a typical poultry diet in which 35% of the diet consisted of DT soybean meal made from MON 87769 soybean, control or one of six different conventional commercial soybean varieties. The SDA content of MON 87769 DT soybean meal is approximately 0.07 percent, resulting in an intake of approximately 100 mg SDA/kg bw/day over the 42 day trial. Monsanto states that no biologically relevant differences in broiler performance, carcass yield, or meat composition were observed between chickens fed diets containing soybean meal obtained from MON 87769 soybean and those fed diets from soybean meal obtained from control or one of the commercial varieties. Monsanto also expects that animal feed containing MON 87769 DT soybean meal will be mixed with other sources of soybean meal, so that the exposure to SDA in animal feed would be significantly lower than reported in this study.
Although Monsanto does not currently plan to feed oil obtained from MON 87769 soybean to animals, Monsanto also provided a published study comparing SDA soybean oil, conventional soybean oil, and fish oil (as a common source of omega-3 fatty acids) added to the diet of chickens at 50 g oil per kilogram of feed. The study authors reported no adverse effects at this level of supplementation, calculated to be 12 g SDA per kg feed or 1.2% by weight. Monsanto also notes that similar or greater levels of SDA from fish oil and other marine oils are currently added to feed of various animal species.
Common or Usual Name of the Oil Product
Monsanto concludes that, based on the intended production of SDA, a new common or usual name is appropriate for the oil from MON 87769 soybean to distinguish this oil from conventional soybean oil as defined in the Food Chemicals Codex (FCC). Monsanto proposed the name "stearidonate soybean oil" for the oil that is produced from MON 87769 soybean.
FDA evaluated Monsanto's submission to determine whether MON 87769 soybean raises any safety or regulatory issues with respect to the intended modification or with respect to the food 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 with food ingredients or soybean meal derived from MON 87769 soybean under the FD&C Act that would require further evaluation at this time.
Monsanto concludes that oil derived from MON 87769 soybean for use as an ingredient in human food is not materially different in safety and other parameters from oils of similar chemical composition produced from other sources and used as an ingredient in human food. Monsanto also concluded that there are no safety concerns from uses of MON 87769 soybeans in human food but noted that the primary human food ingredient derived from this soybean will be the oil due to its altered fatty acid profile. In addition, Monsanto has concluded that meal derived from MON 87769 is as safe for use in animal feed as conventional soybean meal. At this time, based on Monsanto's data and information, the agency considers Monsanto's consultation on MON 87769 soybean to be complete.
Robert I. Merker, Ph.D.
Mary D. Ditto, Ph.D.
 The soybean Sphas1 gene encodes the alpha prime subunit of the beta-conglycinin seed storage protein.
 The soybean Sphas2 gene encodes the alpha subunit of the beta-conglycinin seed storage protein.
 Monsanto also confirmed the (continued) absence of plasmid backbone and T-DNA II sequences over four generations.
 Monsanto notes that Fos-choline 12 is a zwitterionic phospholipid analog detergent. It has structure-stabilizing properties, has been used in NMR studies of membrane bound proteins, and allows refolding of integral membrane proteins.
 Monsanto reports that all chromatographic steps were conducted in a cold room, protease inhibitors were added at crucial steps, and reducing agents and glycerol were added to buffers.
 N-terminal sequence analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); matrix assisted laser desorption ionization – time of flight (MALDI-TOF) mass spectroscopy to generate a tryptic peptide map; immunoblot analysis to establish protein identity through immunoreactivity with specific antibodies; SDS-PAGE to assess apparent molecular weight of the protein; and glycosylation analysis to evaluate potential post-translational modification of the protein.
 Monsanto states that a similar in vitro demonstration of the functional activity of NcΔ15D protein in crude homogenate from immature MON 87769 soybean seed was not successful because soybean contains an endogenous Δ15 desaturase; therefore, the desaturase reaction occurs to some extent in the conventional control soybean seed.
 The proteins used in the studies were extracted from MON 87769 immature seed.
 Monsanto submitted a GRAS notice (GRN 000283) for use of SDA soybean oil in a variety of human foods. FDA responded that it had no questions regarding Monsanto's conclusion that the intended uses of SDA soybean oil are GRAS on September 4, 2009.
 A 99% tolerance interval represents, with 95% confidence, 99% of the values contained in the population of commercial conventional soybean varieties.
 Monsanto used version 3.0 of the ILSI-CCD in its analysis. The database is maintained by ILSI and can be accessed at http://www.cropcomposition.org/.