Biotechnology Consultation Note to the File BNF No. 000121
Return to inventory: Submissions on Bioengineered New Plant Varieties
Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000121
DATE: January 14, 2011
Subject: High Oleic Soybean MON 87705
Keywords: Soybean, Glycine max, glyphosate, cp4 epsps, fatty acid, palmitic acid, oleic acid, linoleic acid, FAD2, FATB, RNA interference, MON 87705, Monsanto, herbicide resistant, modified oil, high oleic, OECD Unique Identifier MON-877Ø5-6
This document summarizes FDA’s evaluation of biotechnology notification file (BNF) No. 000121. In a submission dated October 29, 2009, Monsanto Company (Monsanto) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of the bioengineered soybean MON 87705, which has a fatty acid profile that has been altered from that of conventional soybean varieties. Monsanto provided additional information on December 16, 2009, January 21, 2010, February 15, 2010, March 26, 2010, and June 14, 2010. FDA evaluated the information in Monsanto’s submissions to ensure that regulatory and safety issues regarding human food and animal feed derived from the new plant variety have been resolved.
FDA considered all information provided by Monsanto as well as other information available to the agency. This document is not intended to restate the information provided in the final consultation in its entirety.
The intended effect of the modification in soybean MON 87705 is to produce soybean seeds with decreased levels of palmitic and stearic acids and increased levels of oleic acid, with an associated decrease in linoleic acid. To accomplish this objective, Monsanto used RNA-based suppression of two key endogenous soybean genes [fatty acid acyl carrier protein thioesterase (FATB1-A) and fatty acid desaturase (FAD2-1A)], the gene products of which are involved in the soybean fatty acid biosynthetic pathway. Monsanto states that FATB1-A and FAD2-1A gene segments were assembled in a single suppression cassette under the control of a seed promoter. Monsanto states that transcription of the introduced FATB1-A and FAD2-1A gene segments produces ribonucleic acids (RNAs) with inverted repeats that form double stranded RNA (dsRNA). The dsRNA thus formed suppresses the production of endogenous soybean FATB and FAD2 transcripts via RNA interference. The notifier states that this suppression results in the altered fatty acid profile in soybean MON 87705.
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 (FFDCA).
Genetic Modification and Characterization
The non-transgenic conventional soybean variety A3525 was used as the recipient in the transformation to create soybean MON 87705.
Transformation Vector and Method
Monsanto used Agrobacterium-mediated transformation of soybean meristem tissue to introduce DNA into soybean A3525. The genetic elements introduced into soybean A3525 from transformation vector PV-GMPQ/HT4404 include a single copy of T-DNA I cassette and a single copy of T-DNA II cassette each flanked by insertion border region sequences. T-DNA I contains a partial-suppression cassette, under the control of the 7Sα´ seed promoter, that contains the sense segments of the FAD2-1A intron and the FATB1-A 5´ untranslated region (UTR) and plastid targeting sequence, and a cp4 epsps expression cassette, under the control of the FMV/Tsf1 promoter sequence, that contains the cp4 epsps gene and the CTP2 targeting sequence. T-DNA II contains the 3´ UTR sequence of the H6 gene and a partial-suppression cassette comprised of antisense segments of the FAD2-1A intron and the FATB1-A 5´ UTR and plastid targeting sequence.
Characterization, Inheritance, and Stability of the Introduced DNA
In order to characterize the introduced DNA, Monsanto conducted Southern hybridization analyses, DNA sequencing, genomic comparisons, and bioinformatic analyses. Monsanto states that the results of the Southern hybridization analyses show that soybean MON 87705 contains a single copy of T-DNA I and a single copy of T-DNA II inserted at a single locus in the genome. Monsanto notes that further analysis of soybean MON 87705 for plasmid backbone sequence using Southern hybridization showed that soybean MON 87705 does not contain any detectable backbone sequence from the transformation vector PV-GMPQ/HT4404.
Monsanto states that the results of DNA sequencing analyses complement the Southern hybridization analyses and show that the cassettes within T-DNA I, as well as the cassettes within T-DNA II, are intact.
Monsanto performed open reading frame (ORF) bioinformatic analyses of the junction site between the soybean genomic DNA and the inserted DNA to determine whether insertion of the introduced DNA might have created any ORF that might encode a toxin or allergen. From the bioinformatic analyses, Monsanto concludes that, even in the highly unlikely occurrence of translation of any of these ORFs, the polypeptide products are unlikely to exhibit allergenic, toxic, or otherwise biologically adverse properties.
Monsanto reports that the same hybridization pattern was observed using Southern hybridization analyses of genomic DNA isolated from plants from three successive generations. Thus, Monsanto concludes that the inserted DNA in soybean MON 87705 is stably integrated into the genome.
During the development of soybean MON 87705, Monsanto recorded segregation data to assess the heritability and stability of the inserted coding sequences present in the genome. Based on the chi square analysis of the segregation data from three generations, Monsanto concludes that the inserted coding sequences of soybean MON 87705 show a Mendelian inheritance pattern as a single locus. Monsanto also concludes that the data on trait inheritance, as revealed by chi-square analysis, are consistent with the molecular characterization data on the stability of the insert across generations.
Protein Characterization - CP4 EPSPS
Identity, Function, and Characterization
Monsanto states that the cp4 epsps gene was used as a selectable marker during the transformation of the parental soybean variety. Monsanto also states that the CP4 EPSPS protein expressed in soybean MON 87705 is identical to the CP4 EPSPS protein in other Roundup Ready™ crops and it confers tolerance to glyphosate. The CP4 EPSPS protein expressed in soybean MON 87705 is derived from Agrobacterium sp. strain CP4 and is similar and functionally identical to endogenous plant EPSPS enzymes, but has a much lower affinity for glyphosate. Monsanto characterized soybean MON 87705-produced CP4 EPSPS protein using various analytical techniques1 and demonstrated the protein’s equivalence with Escherichia coli-produced CP4 EPSPS protein. Due to the low levels of CP4 EPSPS protein produced in soybean MON 87705, the E. coli-derived CP4 EPSPS protein was used for safety assessment studies.
The CP4 EPSPS protein levels in soybean MON 87705 were measured in replicated samples of leaf, root, forage, and mature seeds from five field sites using a validated enzyme-linked immunosorbent assay (ELISA). Monsanto reports that the range of CP4 EPSPS protein in soybean MON 87705 leaf, root, forage, and mature seeds was 40 to 1000 micrograms per gram dry weight (μg/g DW). The mean CP4 EPSPS protein concentrations (μg/g DW) in soybean MON 87705 were 200 to 530 for leaf at four vegetative stages, 120 for root, 110 for forage, and 77 for mature seeds.
Assessment of Potential for Allergenicity and Toxicity
Monsanto performed studies to assess the potential for allergenicity and toxicity of the CP4 EPSPS protein.
Monsanto performed a sequence similarity search of the CP4 EPSPS amino acid sequence against the TOX_2009 database (which contains sequences of proteins that may be harmful to human and animal health) using the FASTA algorithm. Monsanto states that the results of the sequence comparisons show that no relevant alignments (alignments that display an E-score of less than 1 x 10-5) were observed between the CP4 EPSPS sequence and sequences in the TOX_2009 database.2 Monsanto thus reports that no structurally relevant similarity exists between the CP4 EPSPS protein 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 up to 572 milligrams CP4 EPSPS protein per kilogram body weight (mg/kg) was administered by gavage to ten male and ten female mice. No treatment-related effects on survival, clinical observations, body weight gain, food consumption, or gross pathology were observed. Monsanto thus concludes that the No Observable Adverse Effect Level (NOAEL) for CP4 EPSPS is 572 mg/kg.
Monsanto evaluated the likelihood that health risks would arise from the acute dietary intake of CP4 EPSPS protein from consumption of food derived from soybean MON 87705. Monsanto calculated the margins of exposure (MOEs) for the general population and for non-nursing infants to be 43,600 and 1,100, respectively. Monsanto concludes that these large MOEs indicate that there are no meaningful risks to human health from dietary exposure to the CP4 EPSPS protein derived from soybean MON 87705.
Monsanto notes that soybean is one of the eight major food allergens. Monsanto compared the amino acid sequence of the CP4 EPSPS protein to the amino acid sequences of known allergens in the Food Allergy Research and Resource Program Database (FARRP_2009) using the FASTA sequence alignment program. None of the identified alignments meet the threshold of greater than or equal to 35% identity over 80 amino acids, and no contiguous stretches of 8 or greater amino acids are shared between the CP4 EPSPS protein and the proteins in the allergen database.3
Monsanto also reports that the CP4 EPSPS protein is rapidly (< 15 seconds) degraded in simulated gastric fluid in vitro.
Based on the results of the allergenicity and toxicity studies, Monsanto concludes that the CP4 EPSPS protein poses a negligible risk to human and animal health upon consumption.
Evidence for Suppression of FATB1-A and FAD2-1A Genes
Monsanto examined the expression of RNA from the endogenous FAD2-1A and FATB1-A genes by Northern hybridization analyses. The results of these analyses show that levels of transcripts of FATB1-A and FAD2-1A in immature seeds of soybean MON 87705 are significantly diminished in comparison with those from the control soybean (A3525). This decreased level of transcription confirms that the endogenous FATB1-A and FAD2-1A genes are functionally suppressed in soybean MON 87705.
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 soybean MON 87705 for these same purposes. Soybean seeds are processed primarily into oil and meal. Commodity 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. Monsanto states that soybean MON 87705 was developed to produce an oil with an unsaturated fatty acid profile more like olive oil or canola oil, while having lower levels of saturated fatty acids like commodity soybean oil. A small fraction of soybean meal is further processed into soy flours and soy proteins for a variety of food uses. Traditional foods prepared from soybeans include tofu, miso, soymilk, tempeh, and soy sauce.
Soybean meal 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. Although soybean and soybean-derived products have some use (~5%) in the manufacture of industrial products including soaps, inks, paints, disinfectants, and biodiesel, the food and feed uses of soybean and its processed products remain the predominant use of soybeans produced in the U.S. and globally.
Scope of Analysis
Monsanto analyzed the composition of forage and seed from soybean MON 87705 and the control soybean A3525 to assess whether the transgenic soybean is similar to non-transgenic soybeans except for the intended change in fatty acid composition. Monsanto also assessed the composition of forage and seed from a total of twenty conventional reference soybean varieties ("reference varieties") grown under the same field conditions as soybeans MON 87705 and A3525. Monsanto used data derived from those 20 reference varieties to generate a 99% tolerance interval for each component.4 Monsanto states that the data illustrate the natural variability in commercially grown soybean varieties. The compositional analysis included key nutrients and antinutrients.
Monsanto states that soybean MON 87705, soybean A3525, and 20 conventional soybean varieties (four different conventional soybean varieties per site) were included in this study. However, one reference variety, damaged by an early frost, was excluded from the study. Seed and forage were obtained from soybeans grown in three replicated plots, planted in a randomized complete block design, at each of five field sites across Chile during the 2007/2008 growing season. Monsanto measured and evaluated 60 components in seed and seven in forage. Monsanto analyzed forage for crude protein, crude fat, moisture, ash, carbohydrates by calculation, acid detergent fiber (ADF), and neutral detergent fiber (NDF). Compositional analysis of seed included crude protein, crude fat, moisture, ash, carbohydrates by calculation, ADF, NDF, fatty acids (26: C8-C24), 18 amino acids, Vitamin E, isoflavones (daidzein, genistein, and glycitein), and antinutrients (phytic acid, trypsin inhibitor, lectin, raffinose, and stachyose). Of the measured components, 17 fatty acids in seed had more than 50% of the observations below the assay limit of quantitation and these components were not statistically analyzed. Thus, statistical analyses were conducted for 50 components (43 in seed and seven in forage).
The data sets were assessed 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 a combination of all five field sites (combined-site). Each individual component for soybean MON 87705 was compared with that of soybean A3525. Statistical significance was declared at 5% level (P ≤ 0.05). When a statistically significant difference in a component was detected between soybeans MON 87705 and A3525 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 soybean MON 87705 mean component values to 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).5
Results of analyses:
Compositional analysis of soybean forage
A significant difference between soybeans MON 87705 and A3525 was detected (P < 0.02) for ash. Monsanto reports that the mean ash level measured for both soybeans MON 87705 and A3525 falls within the 99% tolerance interval calculated by Monsanto for the reference varieties, and within the range of literature values for this component. Thus, Monsanto concludes that the difference in ash content is not biologically significant.
Compositional analysis of soybean seed
For the combined-site analyses, a statistically significant difference between soybeans MON 87705 and A3525 was detected (P < 0.048 and P < 0.043) for arginine and cystine (as % of dry weight (DW)). Monsanto reports that the mean arginine and cystine levels measured for both soybeans MON 87705 and A3525 fall within the 99% tolerance intervals calculated by Monsanto for the reference varieties and within the range of literature values. A significant difference between soybeans MON 87705 and A3525 was also detected (P < 0.001) for crude fat (% DW). Monsanto reports that the mean crude fat levels measured for both soybeans MON 87705 and A3525 fall within the 99% tolerance interval calculated by Monsanto for the reference varieties and within the range of literature values. Monsanto concludes that the small differences in arginine, cystine, and total fat are not considered biologically meaningful for food and feed safety or nutrition.
Intended Compositional Change - Fatty Acids
Monsanto states that soybean MON 87705 was developed to produce soybean oil with higher levels of 18:1 oleic acid, an associated decrease in 18:2 linoleic acid and lower levels of 16:0 palmitic and 18:0 stearic acids through suppression of FAD2 and FATB RNAs. Thus, Monsanto reports that there are statistically significant decreases in palmitic acid (P < 0.001) and linoleic acid (P < 0.001) and an increase in oleic acid (P < 0.001), which are associated with the intended effects of the change in this variety (Table 1). Monsanto states that although stearic acid levels are statistically lower in soybean MON 87705 when compared with soybean A3525, all values for soybeans MON 87705 and A3525 fall within the 99% tolerance interval calculated by Monsanto for the reference varieties and within the range of values in the ILSI database. The three remaining statistically significant differences in the combined-site analysis are 18:3 linolenic, 20:0 arachidic, and 20:1 eicosenoic acids. The decrease in linolenic acid is expected, given that this fatty acid is produced from linoleic acid which is reduced by the suppression of the FAD2 gene. Although arachidic acid levels are statistically lower in soybean MON 87705 when compared with soybean A3525, all values for soybeans MON 87705 and A3525 fall within the 99% tolerance interval calculated by Monsanto for the reference varieties. The mean level of eicosenoic acid in soybean MON 87705 is significantly higher than in soybean A3525 in the combined-site and all five individual-site analyses. However, the absolute magnitude of these differences is small (< 0.18% of total fatty acids). The combined-site mean for eicosenoic acid (0.34% of total fatty acids) is slightly (0.09% of total fatty acids) outside the upper end (0.25% of total FA) of the 99% tolerance interval, but within the values reported in the ILSI-CCD. Monsanto concludes that the small differences in linolenic, arachidic, and eicosenoic acids are not considered biologically meaningful for food and feed safety or nutrition.
|Fatty Acid||Soybean MON 87705[Range]||Control(Soybean A3525)[Range]||Conventional Tolerance Intervala|
|16:0 Palmitic||2.25-2.44||10.51-11.08||7.62, 12.55|
|18:0 Stearic||3.07-3.82||4.24-4.85||2.87, 7.15|
|18:1 Oleic||73.13-79.17||21.41-25.08||18.40, 30.22|
|18:2 Linoleic||7.85-12.42||51.68-53.89||47.75, 56.46|
|aA 99% tolerance interval represents, with 95% confidence, 99% of the values contained in a population of conventional soybean varieties grown at the same location as the test varieties.|
Compositional analysis of processed fractions (meal, oil, protein isolates, and lecithin)
Seed from soybeans MON 87705 and A3525 were collected from two plots at each of two sites across the U.S. during the 2007 growing season. In addition, 12 conventional soybean varieties produced at three locations in separate U.S. field trials in 2007 were included for the generation of a 99% tolerance interval. A subsample of each soybean MON 87705, control soybean (A3525), and reference soybean seed was processed into toasted and defatted (TD) soybean meal, refined bleached and deodorized (RBD) oil, protein isolate, and crude lecithin. Seed and a subsample of the processed products from MON 87705, A3525, and the 12 conventional soybean varieties were analyzed for composition. In all, 27 components were analyzed in meal, 39 in oil, 19 in protein isolates, and 4 in lecithin. The components in soybean meal included moisture, crude protein, crude fat, ash, carbohydrates by calculation, ADF, NDF, amino acids (18), phytic acid, and trypsin inhibitor.
Oil samples were analyzed for fatty acids (38; C8-C24) and vitamin E. Of the measured components, 21 fatty acids in oil had more than 50% of the observations below the assay limit of quantitation and these components were not statistically analyzed. The components in protein isolates included amino acids (18) and moisture. The components in lecithin were L-α-phosphatidic acid, L-α-phosphatidylcholine, L-α-phosphatidylethanolamine, and L-α-phosphatidylinositol.
Analyses of components in the processed product samples (meal, oil, protein isolates, and lecithin) show no statistically significant differences between soybeans MON 87705 and A3525 for 49 of 68 comparisons. In soybean meal, significant differences were observed for alanine (P < 0.019), glycine (P < 0.023), isoleucine (P < 0.006), lysine (P < 0.030), valine (P < 0.003), and NDF (P < 0.016). For these five amino acids, the absolute magnitude of the mean differences from soybean A3525 were small (< 0.1% DW) and the mean values for soybeans MON 87705 and A3525 fall within the 99% tolerance interval for the reference varieties and also within the range of published values for conventional soybean varieties. Monsanto reports that the mean NDF values measured for soybeans MON 87705 and A3525 fall within the 99% tolerance intervals for reference varieties and also within the range of published values for conventional soybean varieties. Of the 17 fatty acids that could be statistically analyzed, significant differences between soybeans MON 87705 and A3525 in RBD oil were observed for 13 fatty acids. Four of the 13 differences were expected as they were due to the intended changes in fatty acid levels as described above. For six of the remaining nine fatty acids (myristic, palmitoleic, margaric, arachidic, eicosenoic, and behenic acids), the absolute magnitude of the differences between the mean values for soybeans MON 87705 and A3525 is less than 0.15% of total fatty acids and the soybean MON 87705 mean values fall within the 99% tolerance intervals for the reference varieties. The three remaining fatty acids [17:1 9cis heptadecenoic acid, 18:2 other trans isomer fatty acids (excluding 9trans,12trans linoleic), and 18:2 6cis,9cis octadecadienoic acid] are minor components in RBD oil (< 0.2% of total fatty acids) and were found in the processed oil and not in the seed prior to being processed. There are no statistically significant differences (P < 0.05) between soybeans MON 87705 and A3525 for the components measured in the protein isolate and crude lecithin fractions.
Monsanto states that the compositional and nutritional assessment supports the conclusion that, except for the intended changes in the levels of specific fatty acids, soybean MON 87705 is compositionally equivalent to conventional soybean varieties. Monsanto concludes that the small, but statistically significant, differences in the components mentioned above are not considered biologically meaningful for food and feed safety or nutrition.
Monsanto conducted a study to determine whether the transformation process may have increased the overall allergenicity of soybean MON 87705 compared to the conventional soybean variety. Using sera from clinically documented, soybean-allergic patients, Monsanto conducted immunoglobulin E (IgE) ELISA studies using protein extracts from soybeans MON 87705 and A3525. Monsanto reports that soybean-specific IgE binding to endogenous allergens in soybeans MON 87705 and A3525 is comparable with the IgE binding to conventional soybeans currently on the market. Therefore, Monsanto concludes that soybean MON 87705 does not pose an increased endogenous soybean allergen risk compared to conventional soybean varieties.
Fatty Acid Intake
Monsanto generated estimates of dietary exposure to various fatty acids from the consumption of oil from soybean MON 87705. Monsanto concludes that based on conservative intake estimates calculated on the assumption that oil from soybean MON 87705 would replace soybean oil currently used in a subset of targeted foods, the intake of oleic acid would increase, while the intakes of palmitic and linoleic acids would decrease and the total fat intake would not be affected. Since humans consume oils from a variety of sources, Monsanto concludes that consumption of oil from soybean MON 87705 will not affect the human diet.
When oil is removed from the soybean, a defatted meal is generated that is used as a protein supplement for animal feed. In an amendment dated January 21, 2010, Monsanto provided examples of poultry and swine diets to demonstrate that the reduced intake of linoleic acid would not lead to a nutritional deficiency for animals consuming feeds containing meal derived from soybean MON 87705. Even though meal derived from soybean MON 87705 would have reduced amounts of linoleic acid, Monsanto states that the animals’ linoleic acid requirements would be easily met by other ingredients that would be included in the diet, such as corn.
Common or Usual Name of the Oil Product
Based on the intended change in fatty acid composition, it is our understanding that Monsanto has concluded that the common or usual name “high oleic soybean oil” is appropriate to distinguish oil from soybean MON 87705 from that of conventional soybean varieties.
Monsanto has concluded that, with the exception of the intended change in fatty acid composition, soybean MON 87705 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 soybean MON 87705 to be complete.
Shayla West-Barnette, Ph.D.
1The analytical techniques discussed in the submission include N-terminal sequence analysis, mass determination of the tryptic peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western hybridization analysis, CP4 EPSPS enzymatic activity analysis, and glycosylation analysis.
2The E-score was set at < 1 x 10-5 to ensure that sequences have sufficient sequence similarity to infer homology.
3These 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.
4A 99% tolerance interval represents, with 95% confidence, 99% of the values contained in the population of conventional soybean varieties.
5Monsanto 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/.