Monsanto Company 09-SY-201U Page 2 of 471 RELEASE OF INFORMATION

Monsanto is submitting the information in this petition for review by the USDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of this information, Monsanto expects that, in adva nce of the release of the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and the opportunity to object to the release of a ny information based on appropriate legal grounds, e.g., responsiveness, c onfidentiality, and/or compet itive concerns. Monsanto understands that a copy of this information may be made available to the public in a reading room and by individual request as pa rt of a public comment period. Except in accordance with the foregoing, Monsanto does not authorize the release, publication or other distribution of this information (incl uding website posting) without Monsanto's prior notice and consent.

© 2009 Monsanto Company. All Rights Reserved.

This document is protected under copyright la w. This document is for use only by the regulatory authority to which it has been submitted by Monsanto Company and only in support of actions requested by Monsanto Co mpany. Any other use of this material, without prior written consent of Monsanto, is strictly prohibited. By submitting this document, Monsanto does not grant any party or entity any right to license or to use the information or intellectual propert y described in this document.Monsanto Company 09-SY-201U Page 4 of 471 EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Serv ice (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility under th e Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7 772) to prevent the introduction and dissemination of plant pests into the U.S. APHIS regulation 7 CFR Part 340.6 provides that an applicant may petition APHIS to ev aluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longe r should be regulated. If APHIS determines that the regu lated article does not present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status in whole for the ne w biotechnology-derived soybean product, MON 87705, any progeny derived from cr osses between MON 87705 and conventional soybean, and any progeny derived from crosses of MON 87705 with other biotechnology-derived soybean that has been granted nonregulated status under 7 CFR

Part 340.

Product Description

Monsanto Company has developed biotec hnology-derived soybean MON 87705 with an improved fatty acid (FA) profile that result s in an oil that has enhanced nutritional characteristics, and improved suitability and stability for food and industrial uses. Currently commodity soybean oi l requires hydrogenation to im prove its stability for use in many foods given its high proportion of polyunsaturated fatty acids. Hydrogenation

results in the formation of trans fatty acids that pose known co ronary health risks. As food companies reformulated foods to replace trans fat-containing hydrog enated oils with healthier alternatives, they have faced challe nges in finding high stability oils that are also relatively low in saturated fat.

Using the extensive information known regard ing the fatty acid biosynthetic pathway in soybean, MON 87705 was developed to selec tively down-regulate, in seed, two key enzymes involved in fatty acid biosynthesis. As a result, MON 87705 soybean oil is lower in saturated fats (6% vs. 15% of to tal fatty acids) and hi gher in monounsaturated 18:1 oleic acid (76% vs. 23% FA), with an associated decrease in the polyunsaturated 18:2 linoleic acid levels ( 10% vs. 53% FA) relative to commodity soybean. Consequently, MON 87705 soybean oil, with improved oxidative stability and lower saturated fats than currently available commodity soybean oil, is suitable for a range of food and industrial applicati ons. In addition, soybean me al derived from MON 87705, which contains very low residual oil, is unchanged in composition relative to commodity soybean meal. MON 87705 also contains the 5-enolpyruvylshikimate-3-phosphate synthase gene derived from Agrobacterium sp . strain CP4 ( cp4 epsps ) encoding the CP4 EPSPS protein that is expressed throughout th e plant conferring tolerance to glyphosate, the active ingredient in the Roundup ® family of agricultural herbicides.

® Roundup is a registered tradem ark of Monsanto Technology LLC.Monsanto Company 09-SY-201U Page 5 of 471 The improved fatty acid prof ile in MON 87705 soybean oil is achieved through the use of endogenous soybean ( Glycine max L.) gene segments configured to suppress FATB and

FAD2 gene expression. MON 87705 contains FATB1-A and FAD2-1A gene segments under the control of a seed promoter, limiting oi l composition modification to this tissue. The assembled gene transcript has an inverted repeat th at produces double stranded RNA (dsRNA) that, via the RNA interference (RNAi) pathway, suppresses endogenous FATB

and FAD2 gene expression, thereby producing the desired fatty acid phenotype (see Figure below).

Schematic of the Soybean Fatty Acid Biosynthetic Pathway and Summary of Modified Fatty Acid Content in Soybean Oil Derived from MON 87705 Panel A: Schematic of the soybean fatty acid biosynthetic pathway Panel B: MON 87705 soybean oil compared to co mmodity soybean oil and other vegetable oils

indicates suppression of endogenous FATB and FAD2 in MON 87705 seeds.

Acyl-acyl carrier protein (ACP) thioesterases (referred to herein as FATB enzymes) are localized in plastids and hydrol yze saturated fatty acids from the ACP-fatty acid moiety. The suppression of FATB results in a decrease in the transport of the saturated fats out of the plastid, thus retaining their availability for desaturation to 18:1 oleic acid (see Figure above). Therefore, suppression of FATB decreases saturated fat content in the oil as well as increasing oleic acid. S ubsequently, this incr eased amount of oleic acid is either delivered to the oil body or endoplasmic retic ulum for further desaturation. Delta-12Monsanto Company 09-SY-201U Page 6 of 471 desaturases (referred to as FAD2 enzymes) de saturate 18:1 oleic acid to 18:2 linoleic acid. The suppression of FAD2 in soybean seed causes reduced de saturation of oleic to linoleic acid thus contributing further to the increase in oleic while reducing linoleic acid content in the oil. Therefore, the overall result of the suppression of these two enzymes is a reduction in saturated 16:0 palmitic and 18:0 stearic fatty acids, an increase in monounsaturated 18:1 oleic acid, and lower le vels of polyunsaturated 18:2 linoleic acid relative to commodity soybean.

The MON 87705 soybean oil improved fatty acid profile provides new options for food companies interested in the formulation of lower saturated fat f oods. Soybean oil is comprised primarily of five major fatty acids: saturated fatty acids 16:0 palmitic and 18:0 stearic acids, monounsaturated 18:1 oleic ac id, and the polyunsaturated fatty acids, 18:2 linoleic, and 18:3 linolenic acids. These fi ve major fatty acids have very different

oxidative stabilities and chemical functiona lities. Conventional soybean oil typically contains 60-65% polyunsaturated fatty acids, mo stly in the form of linoleic acid. This composition makes soybean oil unsuitable for certain food applications since the high concentrations of polyunsaturated fatty acid s in the oil are susceptible to oxidation and degradation at high temperature. Therefore, hydrogenation of soybean oil is necessary to reduce levels of polyunsaturated fatty acids by converting them to more saturated fatty acids resulting in higher stability oil suitabl e for a range of food uses. The hydrogenation process used to reduce polyunsat urated fatty acids and increas e the stability of soybean oil produces trans fatty acids that are linked to incr eased cardiovascular risk due to the elevation of low-density lipoproteins (L DL) and reduced levels of high-density lipoproteins (HDL). Because MON 87705 soybean oil has a reduced level of polyunsaturated fatty acids, it has higher oxidative stabili ty without the need for hydrogenation, while also containing a lower le vel of saturated fats. Saturated fats, notably palmitic acid, have also been shown to contribute to cardiovascular disease and

other chronic diseases. As a result, the reduced satura ted fat levels in MON 87705 soybean oil can also positively impact the goa l of keeping human dietary consumption of saturated fats below 10% of total energy intake 1

. In addition to providing improve d formulation options for food companies, the reduction

in saturated fats and increased oxidative stability of MON 87705 soybean oil increases suitability for biodiesel and other industrial applications. Low saturated fats and high (>70%) oleic acid levels are key attributes for vegetable oils targeted for biodiesel and

industrial uses because of im proved cold weather performance, improved stability, and

reduced nitrous oxide emissions.

Therefore, the fatty acid profile of MON 87705 soybean oil, with an increase in

monounsaturated oleic acid and decreases in saturated fats and polyunsaturated fatty acids (17% vs. 60% FA), significantly im proves the soybean o il functionality and

nutritional value. MON 87705 will be bred w ith current commercial low linolenic acid soybean varieties that will further enhance the oxidative stability of soybean oil.

1 Department of Health and Human Services, Dietary Guidelines for Americans , 2005. www.health.gov/dietaryguidelines [Accessed June 7, 2009]Monsanto Company 09-SY-201U Page 7 of 471 Studies Confirm the Lack of Pl ant Pest Potential of MON 87705

The data and information presented in this petition demonstrate MON 87705 is agronomically, phenotypically, and compositiona lly comparable to conventional soybean with the exception of the intended modifica tions to oil composition and tolerance to glyphosate. Moreover, the da ta presented show MON 87705 is unlikely to pose an increased plant pest risk, including weed iness or adverse environmental impact, compared to conventional soybean. The fo od, feed and environmental safety of MON 87705 was confirmed based on multiple , well established lines of evidence:

1. Soybean is a familiar crop that does not possess any of the attributes commonly associated with weeds, has a history of safe consumption, and serves as an appropriate basis of comparison.

2. A detailed molecular characterization of the inserted DNA demonstrated a single, intact copy of the transgenic insert in a single locus within the soybean genome.

This insert contains the FATB and FAD2 suppression cassette and the cp4 epsps expression cassette.

3. The inverted repeat encoded by the FATB and FAD2 suppression cassette in MON 87705 does not code for any prot ein. The RNA-based suppression of FATB

and FAD2 soybean genes in MON 87705 is mediated by double stranded RNA (dsRNA) molecules. Double stranded RNAs are commonly used by eukaryotes, including plants, for endogenous gene suppression and pose no novel risks from a food, feed or environmental perspective. Nucleic acids, such as RNA, have a long history of safe consumption and ar e considered GRAS by the U.S. Food and Drug Administration.

4. The only introduced protein produced in MON 87705 is CP4 EPSPS. Data confirmed the CP4 EPSPS protein in MON 87705 is unlikely to be a toxin or allergen based on extensive information collected and evaluations performed. The CP4 EPSPS protein in MON 87705 has the same functional and enzymatic activity as the CP4 EPSPS in other Roundup Ready ® crops previously deregulated by USDA, and is structurally homologous to EPSPSs naturally present in other

crops.

5. A compositional assessment confirmed that, except for intended fatty acid changes, MON 87705 seed and forage are co mpositionally equivalent to seed and forage of conventional soybean. M ON 87705 soybean oil does not contain any new fatty acids that are not already present in commod ity soybean oil, and the fatty acid profile of MON 87705 soybean o il is similar to many other commercial oils currently available.

6. An extensive evaluation of MON 87705 phe notypic and agronomic characteristics

and environmental interactions de monstrate MON 87705 shows no increased plant pest risk potential comp ared to conventional soybean.

® Roundup Ready is a registered trad emark of Monsanto Technology LLC.Monsanto Company 09-SY-201U Page 8 of 471 7. An assessment of potential impact to nontarget orga nisms (NTOs) and endangered species indicates MON 87705 is unlikely to have adverse effects on these organisms compared to conventional soybean under normal agricultural practices.

8. An evaluation of MON 87705 on current cu ltivation and management practices for soybean concluded deregulation of MON 87705 will not significantly impact current soybean agronomic practices and land use.

Soybean is a Familiar Crop Lacking Weedy Characteristics

There is a long standing history of safe consumption of conventional soybean and its oil, as soybean is the most prevalently grown o ilseed in the world, with approximately 222.1

million metric tons of harvested seed (MMT) produced in 2007, representing 56% of world oilseed seed production th at year. Soybean is grown as a commercial crop in over 35 countries and domestication of this crop ca n be traced back to approximately 1000 B.C. A major food use for soybean is purifie d oil, for use in margarines, shortenings, cooking, and salad oils.

The commercial Glycine species in the U.S. ( Glycine max L.) does not exhibit weedy

characteristics and is not effective in invadi ng established ecosystems. Soybean is not listed as a weed in major weed references, nor is it present on the lists of noxious weed species distributed by the fe deral government. Soybean does not possess any of the attributes commonly associated with weeds, such as long pers istence of seed in the soil, the ability to disperse, invade, and become a dominant species in new or diverse landscapes, or the ability to compete well with native vege tation. Due to a pronounced lack of dormancy, soybean seed can germinate quickly under adequate temperature and moisture and potentially can gr ow as a volunteer plant. Ho wever, a volunteer plant likely would be killed by frost during au tumn or winter of the year it was produced. If it did become established, a volunteer plant woul d not compete well with the succeeding crop,

and could be controlled readil y by either mechanical or ch emical means. In addition, since wild populations of Glycine species are not known to exis t in the U.S., the potential

does not exist for MON 87705 to outcross to wild or weedy relatives and alter their weediness potential.

The genetic background of MON 87 705 was matched with that of an appropriate control, so the effect of the genetic insertion and th e presence of the CP4 EPSPS protein could be

assessed in an unbiased manner. Sin ce MON 87705 was derived from the A3525 conventional variety, it was deemed appropriate to use the nontransformed A3525 as the control variety because its use would mi nimize the potential bias in subsequent comparative assessments.

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

MON 87705 was produced by Agrobacterium -mediated transformation of soybean with the binary vector PV-GMPQ/ HT4404 that contains two T-DNAs. T-DNA I and T-DNA II both contain DNA segments designed to suppress endogenous FAD2 and FATB genes which encode for two key enzy mes in the soybean fatty acid biosynthetic pathway. TDNA I also contains a cp4 epsps expression cassette. The partial suppression cassette in T-DNA I contains the sense segments of the FAD2-1A intron and FATB1-A 5'Monsanto Company 09-SY-201U Page 9 of 471 untranslated region (UTR) which are under the regulation of the 7S α ' seed promoter. TDNA II contains a partial suppr ession cassette that contains the antisense segment of

FAD2-1A and FATB1-A.

During plant transformation, the two T-DNAs (T-DNA I and T-DNA II, respectively) cointegrated into one locus in the soybean ge nome. The cointegration of the T-DNAs in this configuration creates an insert containing a single cp4 epsps expression cassette and a single FAD2-1A and FATB1-A suppression cassette. The in planta assembled suppression cassette includes an inverted repeat th at results in suppression of endogenous FAD2 and

FATB RNA expression.

Molecular characterization of MON 87705 by Southern blot demonstrates there is one copy of each T-DNA insert within the same locus of integr ation. Backbone sequences from plasmid PV-GMPQ/HT4404 were not de tected in the genome of MON 87705. Additionally, the data confirm the organization and seque nce of the insert, demonstrate the stability of the insert over several gene rations, and that the genomic DNA sequences flanking the 5' and 3' ends of the inse rt are native to the soybean genome.

RNA-Based Suppression Technology in MON 87705 Does not Pose Unique Safety

Risks

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is mediated by dsRNA molecules. Doubl e stranded RNAs are commonly found in eukaryotes, including plants, for endogenous gene suppression and are composed of nucleic acids. Nucleic acids have a long hist ory of safe consumption and are considered GRAS by the U.S. Food and Drug Administra tion. There is no evidence to suggest dietary consumption of RNA is associated wi th toxicity or allergenicity. Moreover, analysis of the DNA segments encoding this dsRNA showed they lack the sequences required for translation initiation and protein synthesis. The production of a protein from the dsRNA encoded by the insert in MON 87705 is highly unlikely. S everal biotechnology-derived plant pr oducts previously deregulated by APHIS were developed using RNA-based suppression mechanisms, incl uding virus-resistant papaya and squash, high oleic soybean, FLAVR SAVR tomatoes, and plum trees resistant to Plum pox virus. Based on this information, it is concluded that the inserted DNA and resulting dsRNA are safe and unlikely to produce a protein or po lypeptide. As a result, the RNA-based suppression technology used in MON 87705 poses no novel risks from a food, feed or environmental perspective.

Data Confirm CP4 EPSPS Protein Safety

A multistep approach was used to characterize and assess the safety of the CP4 EPSPS protein expressed in MON 87705 . This detailed characterization confirms the CP4 EPSPS protein is safe for human and animal consumption. The assessment involved: 1) characterizing the physicochemical and func tional properties of the protein; 2) quantifying protein levels in MON 87705 plant tissues; 3) examining the similarity of the CP4 EPSPS protein to known allergens, toxi ns and other biological ly-active proteins known to have adverse effects on mammals; 4) evaluating the digestib ility of CP4 EPSPS protein in simulated gastrointestinal fl uids; 5) documenting th e history of safe consumption CP4 EPSPS protei n; and 6) investigating potential mammalian toxicity through an oral gavage assay. Results confirm that CP4 EPSPS is expressed in all tissuesMonsanto Company 09-SY-201U Page 10 of 471 collected, including root, forage, seed, and leaf tissues at four developmental stages. CP4 EPSPS has no relevant amino acid sequence sim ilarities with known al lergens, gliadins, glutenins, or protein toxi ns which can have adverse effects on mammals. Studies utilizing the CP4 EPSPS protein revealed that it degrades ra pidly in simulated gastric and

intestinal fluids and mouse acute oral toxic ity evaluations demonstr ate the protein is not acutely toxic and does not cause any adverse e ffect, even at the highest dose tested. The safety assessment supports the conclusion th at dietary exposure to CP4 EPSPS protein derived from MON 87705 poses no meaningful risk to human or animal health.

MON 87705 is Compositionally E quivalent to Conventional Soybean, Except for the Intended Fatty Acid Changes

Detailed compositional and nut ritional comparisons were conducted to assess whether levels of nutrients, anti-nut rients, and key secondary meta bolites in seed and forage derived from MON 87705 are comparable to th ose in a conventional soybean control and several commercially available reference varieties. The analysis included proximates (protein, fat, carbohydrates, fiber, ash, moistu re) in seed and forage, and fatty acids, amino acids, vitamin E, and antinutrients in seed. Composition data were analyzed statistically for all component s and their biological and nu tritional significance were evaluated. Statistical analys es were conducted on data from a combination of all sites (combined-site) and data from each of five individual sites (individual-site). If a significant difference (p<0.05) in an analyte was detect ed between MON 87705 and its

conventional control in the combined-s ite comparison, an analysis including reproducibility across individua l sites, magnitude of differ ences, and comparisons of MON 87705 mean analyte values to the 99% tolerance interval for the population of commercial conventional soybean varieties grown concurrently at the same time and field sites and published values wa s made to assess whether the difference was biologically meaningful from a food and feed safety or nutritional perspective. The compositional analysis confirmed that MON 87705 seed ha d the intended fatty acid composition, while the other components analyzed in MON 87705 s eed and forage were considered to be compositionally equivalent to conventiona l soybean. Moreover, no new fatty acids beyond those presently found in soybean were detected in MON 87705.

Of the nine fatty acids that were analyzed statistically, significant differences (p<0.05) were observed for seven fatty acids in th e combined-site analysis. As intended, MON 87705 seed had significantly (p<0.05) lo wer 16:0 palmitic and 18:0 stearic acid levels (combined saturates 5.7%), higher 18 :1 oleic acid (76.5%) and an associated decrease in 18:2 linoleic acid (10.1%) compared to conventional soybean. Differences in these four fatty acids were consistently obser ved at each of the individual sites and levels fell within the intended fatty ac id ranges. A combined-site statistical difference (p<0.05)

between MON 87705 and the conven tional control was observed in the levels of 18:3

linolenic acid. The decrease in 18:3 linolenic acid is expected given that it is produced

from 18:2 linoleic acid which was reduced by the suppression of the FAD2 gene. Examination of the reproducibility within s ites shows the levels of 18:3 linolenic acid were significantly lower than the soybean contro l in four of five i ndividual-site analyses, with the absolute magnitude of the differences being small (<1.5% tota l FA content). In addition, all the mean levels of 18:3 li nolenic acid in MON 87705 seed from the combined-site and individual-site analyses we re well within the 99% tolerance interval,Monsanto Company 09-SY-201U Page 11 of 471 and therefore these differences are not cons idered biologically relevant compositional changes. Combined-site statistical differences between MON 87705 and the

conventional control were also observed in the levels of two minor fatty acids, 20:0 arachidic acid, and 20:1 eicosenoic acid. Exam ination of the reproduc ibility within sites shows the absolute magnitude of the differen ces for these two minor fatty acids was very small (<0.19% of total FA) and mean values and ranges in MON 87705 seed were within

either the 99% tolerance in terval for the population of the conventional reference varieties or the values reported in Interna tional Life Science Institute-Crop Composition Database (ILSI-CCD) and published literature . No difference was observed between

MON 87705 and the conventional control for th e remaining two minor fatty acids (22:0 behenic acid and 24:0 lignoceric acid) evaluated. Thus, ap art from the intended or expected differences in fatty acid levels, there were no other biologically meaningful

differences in the levels of other fatty acids.

Combined-site analyses of both forage and seed samples for non-fatty acid analytes showed no statistically significant diffe rence (p>0.05) between MON 87705 and the conventional control for 37 of 41 analyte co mparisons. Statistical comparisons between

MON 87705 and the conventional control for the presence of other components showed that three analytes in soybean seed (cystine , arginine and total fa t) and one analyte in forage (ash) were significantly different (p<0.05 ) in the combined-site analysis. For these four analytes where differences were not ed (p<0.05), the absolute magnitude of

differences between MON 87705 and the conven tional soybean control were generally low (<1.1% dw), were not observed consistent ly across individual si tes (individual-site analyses), and mean values for MON 87705 we re within the calculated 99% tolerance interval for the population of c onventional reference va rieties. Harveste d seed and forage analytical component values also were compar able to values reported in the ILSI-CCD and/or published literature, further supporti ng the conclusion that harvested seed and forage from MON 87705 are compositionally equivalent to those of conventional soybean. Therefore, it is concluded that the statistical differences observed are not biologically meaningful.

In addition to the compositional analysis of seed and forage, four soybean processed fractions (refined oil, meal, lecithin, a nd protein isolate) were produced from

MON 87705 and conventional control seed and subjected to compositional analysis in accordance with OECD guidelines. As expected , and consistent with results obtained for seed fatty acid levels, the intended fatty acid changes were observed in the refined oil fraction. As in seed, levels of several other less abundant fatty acids were also significantly different (p<0.05) between th e refined soybean oil fractions from MON 87705 and the conventional soybean cont rol. For these analytes, MON 87705

mean values fell within the 99% tolerance intervals for the reference varieties and/or were comparable to published literature range s for conventional soybean oil. Differences in the levels of two other minor fatty acids , likely formed by spontaneous isomerization during the oil refining process, were also observed. Since these differences are not considered biologically relevant, as they likel y are an artifact of the refining process. Apart from the intended fatty acid changes in the oil, the com position of the soybean processed fractions from MON 87705 is equi valent to the composition of the soybean processed fractions from the conventional soybe an control. Thus, the processed fractionsMonsanto Company 09-SY-201U Page 12 of 471 from MON 87705 are as safe and nutritious as the processed fractions from conventional soybean.

The compositional analysis confirmed that MON 87705 seed had the intended fatty acid profile. In all compositional and nutritional comparisons of MON 87705 to its conventional control where a significant diffe rence (p<0.05) was detected, other than for intended fatty acid changes, an analysis incl uding reproducibility acro ss individual sites, magnitude of differences, and comparisons of mean test analyte values to the 99% tolerance interval and published values, indi cated that differences observed were not biologically meaningful from a food and feed safety or nutritional perspective. Therefore, except for the intended fatty ac id changes, the com positional and nutritional assessment of MON 87705 supports the conc lusion that seed and forage and key processed fractions produced from MON 87705 are compositionally equivalent to those of conventional soybean in acco rdance with OECD guidelines.

MON 87705 Does Not Change Soybean Plan t Pest Potential or Environmental

Interactions

The phenotypic, agronomic, and environmenta l interaction assessment indicates that MON 87705 is comparable to the parental conventional soybean control, A3525, which has background genetics similar to MON 87705, but lacks the introduced traits. Thus, MON 87705 is unlikely to have ch anged plant pest risk poten tial or environm ental impact compared to conventional soybean. An impor tant element in assessing plant pest risk potential and environmental impact of MON 87705 is to compare MON 87705 to conventional soybean. The assessment is ba sed initially on the c oncept of familiarity, which USDA recognizes plays an important ro le in these assessments. Familiarity is based on the fact that the biotechnology-deri ved plant is developed from a conventional plant variety whose biological properties and plant pest potential are known to experts. Familiarity considers the biology of the crop, the introduced tr ait, the receiving environment and the interactions among th ese factors, and provides a basis for comparative risk assessment between a biot echnology-derived plant and its appropriate conventional counterpart. The MON 87705 characteristics asse ssed include: seed dormancy and germination, pollen morphology, a nd symbiont interactions conducted in the laboratory and greenhouse, and plant phe notypic observations and environmental

interaction evaluations c onducted in the field.

Seed dormancy and germination characterization indicated that MON 87705 seed had germination characteristics similar to that of the conventional soybean control. In particular, the lack of hard seed, a well-ac cepted characteristic of weediness affecting seed germination rate and viability, supports a conclusion of no increased weediness potential of MON 87705 compared to c onventional soybean for germination and dormancy characteristics. For pollen charact eristics and symbiont interactions, there were no statistically significant differences (p<0.05) observed for any of the parameters measured, including pollen viability, nodule dr y weight, and shoot total nitrogen. Collectively, these results support the conclu sion that MON 87705 is not likely to exhibit increased weed potential compared to conventional soybean.

The field evaluation of phenotypic, agrono mic, and ecological characteristics of MON 87705 also supports the conclusion that MON 87705 is not likely to pose anMonsanto Company 09-SY-201U Page 13 of 471 increased weed or plant pest potential compared to conventional soybean. The evaluations were conducted at 17 replicated field sites across U.S. soybean production regions. These assessments in cluded 14 plant growth and development characteristics, as well as observations for plant-in sect and plant-diseas e interactions and plant responses to abiotic stressors. The observed phenotypic characteristics were comparable between MON 87705 and the conventional soybean cont rol. Across sites, data show no statistically significant differences betw een MON 87705 and the control for seedling vigor, plant height, lodging, pod shattering, seed mois ture, test weight, or yield. Flower color and plant pubescence data were categori cal and were not statistically analyzed; however, at each site, all plan ts of MON 87705 and the cont rol had purple flowers and pubescence as expected.

Four statistically si gnificant differences (p<0.05) were detected betw een MON 87705 and the control in the combined-site analysis. MON 87705 was lower than the control for early stand count, final stand count, the weight of 100 seeds and flowered approximately one day later than the control. MON 87705 a nd the control were within the same range of plant growth stages for 113 out of the 114 growth stage observations among the sites, and the single different observa tion at one site, was within the range of growth stages

observed for the reference varieties. None of these differences were considered biologically meaningful in terms of increased weed potential.

In an individual site assessment of plant res ponse to abiotic stress, disease damage, and arthropod damage, no differences were obs erved between MON 87705 and the control for 574 of 579 comparisons (including 167 ab iotic stress response, 206 disease damage, and 206 arthropod damage comparis ons) among all observations at the 17 sites. The five

observed differences were in the disease and arthropod damage categories. For each of the five observed differences, the severity of damage in MON 87705 was within the range of the reference soybean va rieties, and were not consistently observed across sites. Therefore, they were not considered biologica lly meaningful in terms of increased weed potential.

In an assessment of pest and beneficial ar thropod abundance, no stat istically significant differences (p> 0.05) were detected between MON 87705 and the control for 95 out of 96 comparisons (including 46 arthropod pest comparisons and 50 beneficial arthropod comparisons) among the collections at the four sites. The single statistically significant

difference was for bean leaf b eetle in a single collection time from a single site, where the

mean abundance value from MO N 87705 plots was lower than the conventiona l control, yet fell within the reference range. The di fferences in pest a nd beneficial arthropod

abundance were not indicative of a consistent plant response associated with the traits and are unlikely to be biologically meaningf ul in terms of plant pest potential or environmental impact of MON 87705 compared to conventional soybean.

The plant phenotypic and ecological interacti on parameters evaluated were used to characterize the plant and its interactions w ith the environment, and to assess the plant pest or weed potential of M ON 87705 compared to the conven tional soybean control. An analysis based on the weight of the evid ence, including reproducib ility, magnitude and direction of a difference, and comparison to reference ranges of the detected differences (p<0.05) found in the evaluation of phe notypic, agronomic and environmental characteristics of MON 87705 compared to th e conventional soybean control supports theMonsanto Company 09-SY-201U Page 14 of 471 conclusion that MON 87705 is not likely to incr ease weed or plant pe st potential or to have a biologically meaningful change in terms of environmenta l impact potential.

MON 87705 Will Not Adversely Affect NTOs or Threatened or Endangered Species

Evaluation of the impacts of MON 87705 on NTOs is a component of th e plant pest risk assessment. Assessment of the exp ected differences between MON 87705 and conventional soybean included the presence of the inserted genes, the expression of the CP4 EPSPS protein, and the improved fatty acid profile in MON 87705 seed. The nature of MON 87705 as a product with no pesticidal activity leads to a conclusion that all exposed organisms are considered to be NTOs. The environmental assessment of

MON 87705 indicates that MON 87705 poses no adverse effect on NTOs or endangered species under normal agricultural practices. The environmental interactions evaluation included data collected in the phenotypic studi es on plant-insect, plant-disease, and plantenvironment interactions. The results of this assessment indicated the presence of the CP4 EPSPS protein and improved fatty acid pr ofile in seed did not unexpectedly alter plant-insect interactions, includ ing beneficial arthropods and in sect pests, or alter disease susceptibility of MON 87705 compar ed to conventional soybean.

The fatty acids present in MON 87705 seed ar e widely prevalent in the environment. Fatty acids play a key role in metabo lic energy storage and as components of phospholipids, which are essential for cellula r membrane formation and function. As natural components of the plant and animal wo rld, these fatty acids are not expected to accumulate, persist or be detrimental to the en vironment. In addition, the improved fatty acid profile in MON 87705 seed results in an o il that is very similar to seed fatty acid profiles in widely cultivated crops, such as canola ( Brassica napus L. and B. campestris L.), thus establishing a history of safe environmental exposure.

The naturally glyphosate-tolera nt EPSPS protein from an Agrobacterium sp., CP4 EPSPS, has been introduced into several conv entional crops, such as soybean, corn and cotton, to provide tolerance to glyphosate, the active ingred ient in the Roundup family of herbicides. There is no toxicity associated with this family of proteins, and since they are ubiquitous in plants and microorganisms, they have a history of safety in the environment. The CP4 EPSPS protein is nontox ic to animals including mammals, birds, and arthropods. Lack of hazard for MON 87705 was established through a combination of biochemical information and experimental data demonstrating the existence of no

reasonable mechanism for harm.

Therefore, the assessment considered per tinent product characteri zation information, information from the protein safety assessments, the history of environmental exposure to these fatty acids, and results from the environmental interaction assessment. Taken together, these data support the conclusi on that MON 87705 is unlikely to have an adverse effect on NTOs or endangered species under normal agricultura l practices in U.S. soybean production.

The potential for MON 87705 to outcross with sexually compatible species, including threatened or endangered species, is un likely in the U.S., since no known wild Glycine species related to cultivated soybean are known to be present in North America. In those world areas where sexually compatible species do exist, the potential to outcross is concluded to be low because soybean is a highly self-pollinated species, with cross-Monsanto Company 09-SY-201U Page 15 of 471 pollination to other soybean varieties occurring at very low frequencies (0 to 6.3%) in adjacent plants. Furthermore, in the rare event when cross-pollination does occur, MON 87705 and its progeny would not have a significant environmental impact, because evaluations have shown the improved fatty acid profile and glyphosate- tolerance traits in MON 87705 have not enhanced weediness or pl ant-pest potential relative to conventional soybean. Therefore, the environmental cons equence of pollen transfer from MON 87705

to other Glycine species is considered negligible.

Deregulation of MON 87705 Will Not Sign ificantly Impact Soybean Agronomic

Practices or Land Use

Soybean fields are typically hi ghly managed agricultural areas that can be expected to be dedicated to crop production for many year s. MON 87705 likely would be used in common rotations on land previ ously used for agricultural purposes. No significant impact would be expected following the introduction of MON 87705 on current cultivation and management practices for s oybean. Except for the intended modification in fatty acid profile and glyphosate tole rance, MON 87705 is no different from conventional soybean in its ag ronomic, phenotypic, ecological, characteristics and has the

same levels of resistance to insects and di seases as current commercial soybean. The introduction of MON 87705 provides growers the means to produce a highly valued oil for use in multiple food and industrial applicat ions. Based on these considerations, there is no apparent potential for significant impact on agronomic practices or land use.

Conclusion

Based on the data and information presented in this petition, it is concluded that MON 87705 is not likely to be a plant pest. Therefore, M onsanto Company requests a

determination from APHIS that MON 87705 and any progeny derived from crosses between MON 87705 and conventional soybean or deregulated biotechnology-derived soybean be granted nonregulated status under 7 CFR Part 340.Monsanto Company 09-SY-201U Page 16 of 471 TABLE OF CONTENTS

RELEASE OF INFORMATION ........................................................................................ 2

CERTIFICATION .............................................................................................................. 3

EXECUTIVE SUMMARY ................................................................................................ 4

TABLE OF CONTENTS .................................................................................................. 16

LIST OF FIGURES .......................................................................................................... 23

ABBREVIATION AND DEFINITIONS ......................................................................... 24

I. RATIONALE FOR THE DEVE LOPMENT OF MON 87705 ..................................... 32

I.A. Basis for the Request for a Determination of Nonregulated Status under

7 CFR Part 340.6............................................................................................. 32

I.B. Rationale for the Development of Nutritionally Improved Soybean

MON 87705 .................................................................................................... 32

I.C. Gene Suppression of FATB and FAD2 and Resulting Fatty Acid Composition of the Seed ................................................................................. 34 I.D. Submissions to Other Regulatory Agencies ................................................... 41

II. THE SOYBEAN FAMILY ......................................................................................... 43

II.A. Soybean as a Crop ........................................................................................... 43

II.B. History of Soybean ......................................................................................... 44

II.C. Taxonomy and Phylogenetics of Soybean ...................................................... 45

II.D. The Genetics of Soybean ................................................................................ 47

II.E. Pollination of Cultivated Soybean .................................................................. 47

II.F. Cultivated Soybean as a Volunteer ................................................................. 48

II.G. Characteristics of th e Recipient Plant ............................................................. 48

II.H. Soybean as a Test System in Product Safety Assessment .............................. 48

III. DESCRIPTION OF THE TRANSFORMATION SYSTEM .................................... 49

IV. GENETIC ELEMENTS ............................................................................................. 51

IV.A. Vector GMPQ/HT4404 ................................................................................... 51

IV.B. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA I) ..... 51

IV.C. The FAD2-1A and FATB1-A segments Sequence (T-DNA I and II) .............. 52

IV.D. Regulatory Sequences ..................................................................................... 52

IV.E. T-DNA Borders .............................................................................................. 52

IV.F. Genetic Elements Outside of the T-DNA Borders ......................................... 53

V. GENETIC ANALYSIS ............................................................................................... 59

V.A. Copy Number of T-DNA I and T-DNA II in MON 87705 ............................ 65

V.B. Southern Blot Analysis to Determine the Presence or Absence of Plasmid PV-GMPQ/HT4404 Backbone ....................................................................... 72

V.C. Organization and Sequence of the Insert and Adjacent Genomic DNA in

MON 87705 .................................................................................................... 76

V.D. Southern Blot Analysis to Examine Inse rt Stability in Multiple

Generations of MON 87705 ............................................................................ 76Monsanto Company 09-SY-201U Page 17 of 471 V.E. Inheritance of the Geneti c Insert in MON 87705 ........................................... 80

V.F. Conclusion of Molecular Characterization ..................................................... 84

VI. CHARACTERIZATION OF THE INTRODUCED CP4 EPSPS PROTEIN IN

MON 87705 .................................................................................................... 85

VI.A. Identity and Function of the CP 4 EPSPS Protein from MON 87705 ............. 85

VI.B. Characterization of the Full Length CP 4 EPSPS Protein from

MON 87705 .................................................................................................... 86

VI.C. Expression Levels of CP4 E PSPS Protein in MON 87705 ............................ 87

VI.D. Assessment of Potential Allergenicity of the CP4 EPSPS Protein from

MON 87705 .................................................................................................... 90

VI.E. Safety Assessment Summary of CP4 EPSPS Protein ..................................... 90

VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENT OF MON 87705 ..... 93

VII.A. Overall Assessment of the Composition of Forage and Seed from MON 87705 Compared to the Conventional Soybean Control ...................... 95 VII.A.1 Intended Changes to Fatt y Acid Levels in MON 87705 Seed ........................ 96 VII.A.2 Fatty Acid Levels in Soybean Seed ................................................................ 97 VII.A.3. Levels of Non-Fatty Acid Nutrients in Soybean Seed .................................... 99 VII.A.4. Naturally Occurring Anti-Nut rient Levels in Soybean Seed ........................ 100 VII.A.5. Proximate and Fiber Levels in Forage .......................................................... 101

VII.B. Compositional Equivalence of MO N 87705 Seed and Forage to Conventional Soybean .................................................................................. 101

VII.C. Compositional Comparison of Processed Fractions from Soybean

Seed of MON 87705 and the Conventional Control ..................................... 112

VII.D Safety and Nutritional Assessment of the Intended Changes in

MON 87705 .................................................................................................. 113

VII.E Safety and Nutrition Assessment Conclusion ............................................... 114

VIII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL INTERACTIONS

ASSESSMENT ............................................................................................. 115

VIII.A. Characteristics Measured for Assessment .................................................... 115

VIII.B. Interpretation of Phenotypic and E nvironmental Interaction Data ............... 117

VIII.C. Interpretation of Detected Differences Criteria ............................................ 118 VIII.D. Phenotypic, Agronomic and Environm ental Interactions

Characteristics ............................................................................................... 119

VIII.E. Overall Conclusions for Phe notypic, Agronomic, and Environmental

Interactions Evaluation ................................................................................. 130

IX. U.S. AGRONOMIC PRACTICES ....................................................................... 131

IX.A. Introduction ................................................................................................... 131 IX.B. Overview of U.S. Soybean Production ......................................................... 132

IX.C. Production Management Considerations ...................................................... 144 IX.D. Weed Management ....................................................................................... 149 IX.E. Management of Insects ................................................................................. 152 IX.F. Management of Diseases and Other Pests .................................................... 152 IX.G. Crop Rotation Pract ices in Soybean ............................................................. 154 IX.H. Soybean Volunteer Management .................................................................. 166 IX.I. Stewardship of MON 87705 ......................................................................... 168Monsanto Company 09-SY-201U Page 18 of 471 IX.J. Impact of the Introduction of MON 87705 on Agricultural Practices .......... 169

X. ENVIRONMENTAL CONSEQUENCES AND IMPACT ON AGRONOMIC

PRACTICES ................................................................................................. 170

X.A. Introduction ................................................................................................... 170 X.B. Plant Pest Assessment of the MON 87705 Insert and Expressed

Substances ..................................................................................................... 171

X.C. Weediness Potential of MON 87705 ............................................................ 176

X.D. Potential for Pollen mediated Gene Flow ..................................................... 177 X.E. Summary of Plan t Pest Assessments ............................................................ 179

XI. ADVERSE CONSEQUENCES OF INTRODUCTION ....................................... 182

REFERENCES .............................................................................................................. 183

Appendix A. USDA Notification ................................................................................... 200

Appendix B. Materials and Methods Used for Molecular Analyses of

MON 87705 .................................................................................................. 203

Appendix C. Materials, Methods and Results fo r Characterization of CP4 EPSPS

Protein Produced in MON 87705 ................................................................. 208

Appendix D. Materials and Methods Used for the Analysis of the Levels of CP4

EPSPS Protein in MON 87705 ..................................................................... 228

Appendix E. Materials and Methods Used for Co mpositional Analysis of MON 87705 Soybean Seed, Forage and Processed Fractions ...................... 231

Appendix F. Materials and Methods for Seed Dormancy and Germination Analyses of MON 87705 .............................................................................. 335

Appendix G. Material, Methods and Individual Site Results from Phenotypic, Agronomic and Ecological Interac tions Analyses of MON 87705 .............. 341

Appendix H. Materials and Methods for Pollen Mo rphology and Viability

Evaluation ..................................................................................................... 363

Appendix I. Materials and Methods for Symbiont Evaluation ................................ 365

Appendix J. Materials and Methods for Analysis of FAD2-1A and FATB1-A

RNA levels in MON 87705 immature seeds ................................................ 367

Appendix K. Manufacturing Process for Soybean .................................................... 370

Appendix L. Glyphosate Resistance ......................................................................... 372

Appendix M. Nutritional Impact Evaluati on of MON 87705 Soybean Oil ............... 390

Appendix N. Glyphosate-Tolerant Crops : Potential Impacts on Land Use and Agricultural Practices .................................................................................... 397

Appendix O. Potential Impact of Glyphosate on Human Health and the

Environment .................................................................................................. 414

Appendix P. Petitioner’s E nvironmental Assessment .............................................. 442Monsanto Company 09-SY-201U Page 19 of 471 LIST OF TABLES

Table I-1. Summary of Product Concept Fa tty Acid Levels for Test (MON 87705) vs. the Conventional Cont rol (A3525) and

Commercial Tolerance Interval ................................................................ 41

Table II-1. World Soybean Production in 2007/2008 ................................................. 43 Table II-2. List of Species in the Genus Glycine Willd., 2n Chromosome Number, Genome Symbol , and Distribution ............................................ 46

Table IV-1. Summary of Genetic Elements in Plasmid Vector PV- GMPQ/HT4404 ................................................................................. 56

Table V-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restric tion Enzymes Used in MON 87705 Analysis..................................................................................................... 62

Table V-2. Summary of Genetic Elements in MON 87705 ........................................ 63

Table V-3. Segregation of the H6 3’UTR Ge ne During the Development of

MON 87705 .............................................................................................. 83

Table VI-1. Tissues collected for MON 87705 ............................................................ 88 Table VI-2. Summary of CP4 EPSPS Protein Levels in Leaf, Seed, Root, and Forage Tissues fr om MON 87705 Grown in 2007/2008 Chile

Field Trials ................................................................................................ 89

Table VII-1. Summary of Intended Changes in Fatty Acid Levels for MON 87705 vs. the Conventional Soybean Control (A3525) in the Combined-Site Analysis ........................................................................... 97

Table VII-2. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON 87705 vs. the Conventional Control (A3525) and Commercial Re ference Varieties ....................................... 102

Table VIII-1. Phenotypic, Agronomic and Environm ental Interaction

Characteristics Evaluated in U.S. Fi eld Trials or Greenhouse Studies ... 116

Table VIII-2. Germination Characteristics of MON 87705 and A3525 ........................ 121

Table VIII-3. Field Phenotypic Evaluation Site s for MON 87705 during 2007 ........... 124 Table VIII-4. Plant Growth and Development Data ac ross 17 Locations during 2007 ............................................................................................. 125

Table VIII-5. Pollen Grain Diamet er and Viability Analyses ....................................... 128 Table IX-1. Soybean Production in the U.S., 1999 – 2008 ........................................ 134 Table IX-2. U.S. Soybean Produc tion by Region and State in 2008 .......................... 135 Table IX-3. U.S. Soybean Productio n Costs and Returns in 2006 ............................. 136

Table IX-4. Common Weeds in Soybean Produc tion: Midwest Region ................... 150

Table IX-5. Common Weeds in Soybean Producti on: Mid-South Region ............... 150

Table IX-6. Common Weeds in Soybean Production: Eastern Coastal Region ........ 151

Table IX-7. Rotational Practices in th e U.S. Following Soybean Production ............ 157

Table IX-8. Rotational Pr actices Following Soybean Production in the Midwest Region ..................................................................................................... 158

Table IX-9. Rotational Practices Fo llowing Soybean Production in the Mid-South Region ................................................................................... 162

Table IX-10. Rotational Practices Follow ing Soybean Production in the Eastern Coastal Region ........................................................................................ 164Monsanto Company 09-SY-201U Page 20 of 471 Table IX-11. Ratings for Control of Volunteer Gly phosate-Tolerant Soybean in Labeled Rotational Crops ................................................................... 167 Table X-1. Summary of Published Lite rature on Soybean Cross Pollination ........... 180 Table A-1. USDA Notifications Approved for M ON 87705 and Status of

Trials Conducted under These Notifications .......................................... 201 Table C-1. Molecular Weight Diff erence Between the MON 87705- and

E. coli - produced CP4 EPSPS Proteins................................................... 215 Table C-2. Comparison of Immunoreactiv e Signal between MON 87705-

Produced and E. coli - produced CP4 EPSPS Proteins ............................ 219 Table C-3. Summary of th e Tryptic Masses Identifie d for the MON 87705- Produced CP4 EPSPS Using MALDI-TOF MS. .................................... 221 Table C-4. N-Terminal Sequence of the MON 87705-produced CP4 EPSPS. ........ 223

Table C-5. CP4 EPSPS Functional Assay ................................................................ 226

Table D-1. Protein Extraction Methods for Tissue Samples ..................................... 229 Table E-1. Statistical Summary of Site CdT Soybean Forage Fiber and

Proximate Content for MON 87705 vs . the Conventional Control (A3525) ................................................................................................... 242 Table E-2. Statistical Summary of Site CdT Soyb ean Seed Amino Acid, Fatty Acid, Fiber, Proximate, V itamin, Antinutrient and Isoflavone

Content for MON 87705 vs. the C onventional Control (A3525) ........... 244 Table E-3. Statistical Summary of Site M EL Soybean Forage Fiber and

Proximate Content for MON 87705 vs . the Conventional Control (A3525) ................................................................................................... 252 Table E-4. Statistical Summary of Site MEL Soyb ean Seed Amino Acid, Fatty Acid, Fiber, Proximate, V itamin, Antinutrient and Isoflavone

Content for MON 87705 vs. the C onventional Control (A3525) ........... 254 Table E-5. Statistical Summary of Site QUI Soybean Forage Fiber and

Proximate Content for MON 87705 vs. the Conventional Control (A3525) ................................................................................................... 262 Table E-6. Statistical Summary of Site QUI Soyb ean Seed Amino Acid, Fatty Acid, Fiber, Proximate, V itamin, Antinutrient and Isoflavone

Content for MON 87705 vs. the C onventional Control (A3525) ........... 264 Table E-7. Statistical Summary of Site R AN Soybean Forage Fiber and

Proximate Content for MON 87705 vs . the Conventional Control (A3525) ................................................................................................... 272 Table E-8. Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, V itamin, Antinutrient and Isoflavone

Content for MON 87705 vs. the C onventional Control (A3525) ........... 274 Table E-9. Statistical Summary of Site SFR Soybean Forage Fiber and

Proximate Content for MON 87705 vs . the Conventional Control (A3525) ................................................................................................... 282 Table E-10. Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, V itamin, Antinutrient and Isoflavone

Content for MON 87705 vs. the C onventional Control (A3525) ........... 284Monsanto Company 09-SY-201U Page 21 of 471 Table E-11. Statistical Summary of Comb ined Site Soybean Forage Fiber and

Proximate Content for MON 87705 vs . the Conventional Control (A3525) ................................................................................................... 292 Table E-12. Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, V itamin, Antinutrient and Isoflavone

Content for MON 87705 vs. the C onventional Control (A3525) ........... 294

Table E-13. Literature and ILSI- CCD Ranges for Components in Soybean Forage

and Seed .................................................................................................. 303 Table E-14. Estimation of 17:1 9c Heptadecenoic Acid Intake in Selected Foods

and MON 87705 ..................................................................................... 308 Table E-15. Summary of Differences (p <0.05) for the Comparison of Soybean Processed Fraction Component Levels for MON 87705 vs. the Conventional Control (A3525) a nd Reference Varieties ........................ 311 Table E-16. Comparison of Amino Acid, Fibe r, Proximate, and Antinutrient

Content from MON 87705 and Conven tional Control (A3525) Soybean Meal Processed from a 2007 U.S. Field Production ................ 320 Table E-17. Comparison of Fatty Acid s and Vitamin E from MON 87705 and Conventional Control (A3525) Soybean RBD Oil Processed from the

2007 U.S. Field Production ..................................................................... 324 Table E-18. Comparison of Amino Acid s and Moisture from MON 87705 and Conventional Control (A3525) Soybean Pr otein Isolate Processed

from a 2007 U.S. Field Production ......................................................... 327 Table E-19. Comparison of Phosphatides from MON 87705 and Conventional Control (A3525) Soybean Lecithin Processed from a 2007 U.S. Field

Production ............................................................................................... 330 Table E-20. Literature Ranges for Components in Soybean Meal ............................. 331 Table E-21. Literature Ranges for Com ponents in Soybean Protein Isolate .............. 332 Table E-22. Literature Ranges for Components in Soybean Lecithin ........................ 332 Table E-23. Literature Ranges for Components in Soybean Oil ................................ 333

Table F-1. Starting Seed of MON 87705, Cont rol and Commercial Reference Soybean Varieties Used in Dormancy Assessment ................................ 337 Table F-2. Comparison of MON 87705 to the Control for Dormancy and

Germination Characteristics .................................................................... 338

Table G-1. Starting Seed for Phenotypic Assessments ............................................. 342

Table G-2. Field and Planting Information ............................................................... 343 Table G-3. Phenotypic Comp arison of MON 87705 to the Conventional Soybean Control within Each Site .......................................................... 349

Table G-4. Growth Stage Monitoring of M ON 87705, the Conventional Soybean Control, and the Reference Soybean Varieties ....................................... 352

Table G-5. Abiotic Stressor Evaluation Us ing Observational Severity Scale for MON 87705 and the Conventional Soybean Control ............................. 356 Table G-6. Disease Damage Evaluations Using an Observational Severity Scale for MON 87705 and the Conventional Soybean Control .............. 357 Table G-7. Arthropod Damage Eval uated Using an Observational Severity Scale for MON 87705 and the Conventional Soybean Control .............. 358Monsanto Company 09-SY-201U Page 22 of 471 Table G-8. Abundance of Pest Arthropods in Be at Sheet Samples Collected from MON 87705, the Conventional Soyb ean Control, and the Reference Soybean Varieties .................................................................. 359

Table G-9. Abundance of Bene ficial Arthropods in Beat Sheet Samples Collected from MON 87705, the Conventional Soybean Control, and the Reference Soybean Varieties ..................................................... 361

Table I-1 Starting seed of MON 87705, Cont rol, and Commercial Soybean Reference Varieties Used in the Symbiont Evaluation ........................... 365 Table L-1. U.S. Glyphosate Re sistant Weeds through June 2009 ............................ 377 Table L-2. Management Reccomendati ons for Control of Glyphosate Resistant

Weeds ...................................................................................................... 382 Table M-1. Fatty Acid Profile for MON 87705 Used in the Intake Estimations ....... 391 Table M-2. Mean Per Capita Fatty Acid Intake from Target Foods Pre and Post MON 87705 Replacement, U.S.population ............................................ 392

Table M-3. 90 th Percentile Per User Fatty Ac id Intake from Target foods, Pre and Post MON 87705 Replacement, U.S.Population ....................... 392 Table M-4. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, U.S.Population (g/day)............................................................................ 393 Table M-5. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, U.S.Population (% en) ............................................................................ 393 Table M-6. Mean Per Capita a nd 90th Percentile Per User Fatty Acid Intake from Total Diet, Post MON 87705, Age and Gender Groups (g/day) .... 394 Table N-1. Herbicide-Tolerant Crops Unde rgoing Review or Pr eviously Deregulated by USDA-APHIS. ................................................................................... 397

Table O-1. Established Glyphosate Tolerances Pr ior to Glyphosate-tolerant Crops (1993) ...................................................................................................... 420 Table O-3. Aggregate Exposur e Assessment for Glyphosate ................................... 422 Table P-1. Deregulated or Submitted Biotechno logy-derived Soybean

Products with Altered Oil Profiles .......................................................... 445 Table P-2. Organic and Conventio nal Soybean Seed Sources ................................. 453 Table P-3. Comparison of Fatty Ac id Profiles from Plant Sources .......................... 458 Table P-5. Deregulated or Submitted Biotechno logy-derived Soybean

Products................................................................................................... 465Monsanto Company 09-SY-201U Page 23 of 471 LIST OF FIGURES

Figure I-1. Schematic of the Soybean Fatty Acid Biosynthetic Pathway ................... 38 Figure I-2. Northern Blot Analysis of FAD2-1A RNA Level in MON 87705 ........... 39 Figure I-3. Northern Blot Analysis of FATB1-A RNA Level in MON 87705............ 40 Figure III-1. Schematic of the Development of MON 87705 ....................................... 50 Figure IV-1. Circular Map of Plasmid PV-GMPQ/HT4404 showing probes 1-6 ......... 54 Figure IV-2 Plasmid Map of Vector PV-GMP Q/HT4404 Showing Probes 7-10 ........ 55 Figure IV-3. Deduced Amino Acid Sequence of the Matu re CP4 EPSPS Protein in PV-GMPQ/HT4404 .............................................................................. 58 Figure V-1. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87705 ........................................................................ 61 Figure V-2. Southern Blot Analysis of M ON 87705: Probes 1, 4, and 6 .................... 69 Figure V-3. Southern Blot Analysis of MON 87705: Probes 2 and 5 ......................... 70 Figure V-4. Southern Blot Analysis of of MON 87705: Probe 3 ................................ 71 Figure V-5. Southern Blot Analysis of MON 87705:PV-GMPQ/HT4404

Backbone Probes 7 and 9 .......................................................................... 74 Figure V-6. Southern Blot Analysis of MON 87705: PV-GMPQ/HT4404

Backbone Probes 8 and 10 ........................................................................ 75 Figure V-7. Breeding History of MON 87705 ............................................................. 78 Figure V-8. Generational Stability of M ON 87705: Probes 1 and 6 ............................ 79 Figure V-9. Breeding Path for Generating Se gregation Data MON 87705 ................. 82 Figure VIII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods ............................................................................ 118 Figure B-1. Overlapping PCR Analysis acro ss the Insert in MON 87705 ................ 207 Figure C-1. Molecular Weight and Purity Analysis of the MON 87705-

Produced CP4 EPSPS Protein ................................................................. 216 Figure C-2. Western Blot Analysis of MON 87705-produced and E. coli - produced

CP4 EPSPS Proteins ............................................................................... 218 Figure C-3. MALDI-TOF MS Coverage Map of the MON 87705-Produced CP4

EPSPS. .................................................................................................... 222 Figure C- 4. Glycosylation Analysis of th e MON 87705-Produced CP4 EPSPS

Protein ..................................................................................................... 225 Figure K-1. Schematic of Manufactur ing Process for Soybean Oil. .......................... 370 Figure L-1. Weed Resistance to Vari ous Herbicide Chemical Families ................... 375 Figure N-1. Total Acres of Canola Relative to Acres of Glyphosate-tolerant Varieties planted. .................................................................................... 399 Figure N-2. Total Acres of Corn and Soyb ean Relative to Acres of Herbicidetolerant (HT) Vari eties Planted. .............................................................. 400 Figure N-3. Total Acres of Cotton Relative to Acres of Herbicide-tolerant (HT)

Varieties Planted. .................................................................................... 401 Figure N-4. Percentage of Ce rtified Organic Acres for Cor n, Cotton, and Soybean. .................................................................................................. 403 Figure O-1. Tier I Endangered Species Assessment .................................................. 428 Figure P-1. Harvested U.S. Acres of Canola, Sunflower and Soybean ..................... 448Monsanto Company 09-SY-201U Page 24 of 471 ABBREVIATIONS AND DEFINITIONS ∗

~ Approximately 2T-DNA Plasmid vector containing two separate T-DNA regions each surrounded by left and right bor ders of the Ti plasmid

7S α ' 3’ region of the Sphas1 gene of Glycine max encoding the 7S α ’ seed storage protein, β -conglycinin, including 35 nucleotides of the carboxylterminal β -conglycinin coding region with the termination codon and the polyadenylation sequence

35S Enhancer sequences from the promoter of the Figwort Mosaic virus (FMV) 35S RNA

aadA Bacterial promoter and coding sequence for an aminoglycosidemodifying enzyme, 3'(9)-O-nucle otidyltransferase from the transposon Tn7

AA Amino acid

ACP Acyl carrier protein

AD_2009 Allergen gliadin and glut en proteins sequence database

ADF Acid detergent fiber

AI Adequate Intake

ANOVA Analysis of Variance

AOAC Association of Analytical Chemists

AOCS American Oil Chemists Society

AOSA Association of Offi cial Seed Analysts

AOSCA Association of Official Seed Certifying Agencies

APHIS Animal and Plant Health Inspection Service

APS Analytical protein standard

ASA America Soybean Association

bp Base pair

B. japonicum Bradyrhizobium japonicum

B-Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA

B-Right Border DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA

BSA Bovine serum albumin

oC Degree Celsius

CAST Council for Agricultural Science and Technology, USDA

CBI Confidential business information

∗ Note: Standard abbreviations, e.g., units of measure, are used according to the format described in

‘Instructions to Authors’ in the Journal of Biological Chemistry

.Monsanto Company 09-SY-201U Page 25 of 471 CdT Calera de Tango Maipo Province, Chile

CES Cooperative Extension Services

CFIA Canadian Food Inspection Agency

CFR Code of Federal Regulations

CI Confidence interval

COA Certificate of analysis

COC Chain of custody

CP(A) Buffer Containing 50 mM MES, pH 5.8, 10% glycerol (v:v), 1 mM

benzamidine-HCl and 1 mM DTT

CP4 Agrobacterium sp strain CP4

cp4 epsps coding sequence for the CP4 EPSPS Protein

CP4 EPSPS 5-enolpyruvylshikimat e-3-phosphate synthase from Agrobacterium sp., strain CP4

CPB Cartagena Protocol on Biosafety

cpm Counts per minute

CS- cp4 epsps Codon modified coding sequence of the aro A gene from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein

CS- rop Coding sequence for repressor of primer protein for maintenance of plasmid copy number in E. coli

. CSFII Continuing Survey of Food Intakes by Individuals

CTAB Hexadecyltrimethylammonium bromide

CTP Chloroplast transit peptide

α -Cyano α -cyano-4-hydroxycinnamic acid

Da Dalton

DAP Days after planting

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

DEEM-FCID Dietary exposure evalua tion model-food commodity intake

database

DHB 2,5-Dihydroxybenzoic Acid

DNA Deoxyribonucleic acid

dNTP Deoxynucleotide triphosphate

dsRNA Double-stranded RNA

DTT Dithiothreitol

DW Dry weight

DWCF Dry weight conversion factor

dwt Dry weight of tissue

ECL Enhanced chemiluminescence

E. coli Escherichia coliMonsanto Company 09-SY-201U Page 26 of 471 EDTA Ethylenediaminetetraacetic acid

EFSA European Food Safety Authority

ELISA Enzyme-linked immunosorbent assay

EPA U.S. Environmental Protection Agency

EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase

E -Score Expectation score

EU European Union

FA Fatty acid

FAARP Food Allergy Research a nd Resource Program Database

FAD2 Glycine max gene for Δ -12 desaturase

FAD2-1A The Glycine max FAD2-1A gene encoding the delta-12 desaturase

FAD2-1A P Partial sequence from intron #1 of the Glycine max FAD2-1A gene encoding the delta-12 desaturase which forms part of the suppression cassette

FAME fatty acid methyl ester

FASTA Algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences

FATB Glycine max gene for Palmitoyl-ACP Thioesterase

FATB1-A The Glycine max FATB1-A gene encoding the palmitoyl acyl carrier protein thioesterase

FATB1-A P Partial sequence from the 5' unt ranslated region and the plastid targeting sequence from Glycine max FATB1-A gene that encodes the palmitoyl acyl carrier protein th ioesterase which forms part of the suppression cassette

FDA U.S. Food and Drug Administration

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FMV Figwort mosaic virus

FONSI Finding of No Significant Impact

FW Fresh weight

fwt Fresh weight of tissue

GenBank A public genetic database maintained by the National Center for Biotechnology Information at the National Institutes of Health, Bethesda, MD, USA

GI Gene sequence identification number

GLP Good Laboratory Practice

GLP-T GLP Technologies

GRAS Generarlly Recognized As Safe

GRR Monsanto Company Guidelines for Keeping Research Records

HCl Hydrochloric AcidMonsanto Company 09-SY-201U Page 27 of 471 HDL High-density lipoprotein

HRP Horseradish peroxidase

H.U. hemagglutinating unit

IDP Identity preserved

IgG Immunoglobulin G

ILSI International Life Sciences Institute

ILSI-CCD International Life Sciences Institute-Crop Co mposition Database

ISO International Organiza tion for Standardization

I- Tsf1 Intron from the Tsf1 gene of Aradidopsis thaliana encoding elongation factor EF-1 alpha

kb Kilo bases

kDa Kilodaltons

KCl Potassium Chloride

LB Laemmli buffer

LDL Low-density lipoprotein

Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA

LOD Limit of detection

LOQ Limit of quantitation

L- Tsf1 5 ′ untranslated leader (exon1) from the Tsf1 gene of Aradidopsis

thaliana encoding elongation factor EF-1 alpha

MAFF Ministry of Agriculture, Forestry and Fisheries, Japan

MALDI-TOF Matrix-assisted laser de sorption/ionization time-of-flight

MALDI-TOF MS Matrix-assisted laser de sorption/ionization time-of-flight mass

spectrometry
MEEC Maximum expected environmental concentration

MEL Melipilla, Melipilla Province, Chile

MES 2-[N-Morpholino] Ethanesulfonic Acid

MH + Protonated mass ion

MHLW Ministry of Health, Labor and Welfare, Japan

mM Millimolar

MMT Million metric tones

MOE Margin of exposure

MRMP Monsanto Resistance Management Plan

MS Mass Spectrometry

MSL Monsanto Scientific Literature

MTSA Monsanto Technology Stewardship Agreement

MUFA Monounsaturated fatty acid

MW Molecular weightMonsanto Company 09-SY-201U Page 28 of 471 MWCO Molecular weight cutoff

MWM Molecular weight marker

N/A Not applicable

NCBI National Center of Biotec hnology Information at the National Institutes of Health, Bethesda, MD, USA

NDF Neutral detergent fiber

NEPA National Environmental Policy Act

NFDM Non-fat dried milk

NHANES National Health and Nutrition Examination Survey

NIST National Institute of Standards and Technology

NMWC Nominal molecular weight cut-off

NOAEL No observed adverse effect level

NOP National organic program

nt Nucleotide

NTO(s) Nontarget organism(s)

OECD Organization for Economic Co-operation and Development

OR Origin of replication

OR- ori-PBR322 Origin of replication from pBR 322 for maintenance of plasmid in

E. coli

OR- ori-V Origin of replication for Agrobacterium derived from the broad host range plasmid RK2

OSL Overseason leaf

P- 7S α ′ Promoter and leader from the Sphas1 gene of Glycine max encoding beta-conglycinin storage protein (alpha ′ -bcsp) that directs transcription in seed

PAGE Polyacrylamid e gel electrophoresis

PBS Phosphate buffered saline

PBST Phosphate buffered saline containing 0.05% (v/v) Tween-20

PCR Polymerase chain reaction

PEG Polyethylene glycol

PEP Phosphoenolpyruvate

P- FMV/Tsf1 Chimeric promoter consisting of enhancer sequences from the promoter of Figwort Mosaic Virus (FMV) 35S RNA combined with promoter from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 alpha

P i Inorganic Phosphate

PIP Plant-incorporated protectant

PMSF Phenylmethanesulfonyl fluoride

polyA + RNA polyA enriched RNA

PPA Plant Protection ActMonsanto Company 09-SY-201U Page 29 of 471 PRESS predicted residual sums of squares

PS(A) Buffer Containing 50 mM Tr is-HCl, pH 7.5, 1 mM DTT, 10% glycerol (v:v), 1.5 M (NH 4 )2SO 4 PRT_2009 A protein sequence database derived from GenBank release 169

PTH Phenylthiohydantoin

PUFA Polyunsaturated fatty acid

PVDF Polyvinylidene difluoride

PVPP Polyvinylpolypyrrolidone

PV-GMPQ/HT4404 Plasmid used to transform the soybean genome to produce

MON 87705

QS(A) Buffer Containing 50 mM Tr is-HCl, pH 7.5, 1 mM EDTA, 1 mM benzamidine-HCl, 4 mM DTT)

QAU quality assurance unit

QUI Quilapilun, Cachapoal Province, Chile

RAN Rancagua, Cachapoal Province, Chile

RBD refined, bleached, and deodorized

RCB Randomized complete block

Right Border DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA

RK2 Broad host range plasmid of In c-P1 originally isolated in Klebsiella pneumonia

RNAi RNA interference

rop Coding sequence for repressor of primer protein for maintenance of plasmid copy number in E. coli

S3P Shikimate-3-phosphate

SAP Shrimp Alkaline Phosphatase

SAS Statistical Analysis System

SCN Soybean cyst nematode

SD Standard deviation

SDS Sodium dodecyl sulfate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE Standard error

SFR San Fernando, Colchagua Province, Chile

SGF Simulated gastric fluid

SIF Simulated intestinal fluid

SOP Standard operating procedure

T/C/R Test/Control/Reference

TD toasted and defatted

TDF Total dietary fiberMonsanto Company 09-SY-201U Page 30 of 471 T-DNA I Transfer DNA I

T-DNA II Transfer DNA II

T-DNA Transferred DNA

T- E9 3 ′ untranslated region of the pea rbcS2 gene which functions to direct polyadenylation of the mRNA

TFA Trans fatty acid(s)

T- H6 3 ′ untranslated sequence of the H6 gene from Gossypium

barbadense encoding a protein involve d in secondary cell wall

TES T hreatened or endangered species
TIU Trypsin inhibitor units

TMB 3,3’,5,5’-tetramethylbenzidene

TOX_2009 Toxin protein sequence database

Tris Tris (hydroxymethyl) aminomethane

tRNA Transfer RNA

TS- CTP2 Targeting sequence from the gene shk G encoding the transit peptide region of Arabidopsis thaliana EPSPS that functions to direct transport of the CP4 E PSPS protein to the chloroplasts

TSSP Tissue-specific site pool

TUG Technology Use Guide

Tween-20 Polyoxyethylenesorbitan monolaurate

Tz Tetrazolium

U Unit (of enzyme activity)

U.S. United States of America

USDA United States Department of Agriculture

USDA-APHIS U.S. Department of Agri culture – Animal and Plant Health Inspection Service

USDA-ARS United States Department of Agriculture – Agricultural Research

Service

USDA-ERS U.S. Department of Agri culture – Economic Research Service

USDA-GRIN United States Department of Agriculture – Germplasm Resources

Information Network

USDA-NASS U.S. Department of Agricult ure – National Agricultural Statistics

Service

USDA-NSHS U.S. Department of Agricu lture – National Seed Health System

USFWS U.S. Fish and Wildlife Service

UTR Untranslated region

V Volts

VOI Verification of Identity

WPEP Weed Performance Evaluation Program v/v Volume per volumeMonsanto Company 09-SY-201U Page 31 of 471 wt weight w/v Weight per volumeMonsanto Company 09-SY-201U Page 32 of 471 I. RATIONALE FOR THE DE VELOPMENT OF MON 87705

I.A. Basis for the Request for a Determ ination of Nonregulated Status under

7 CFR Part 340.6

The Animal and Plant Health Inspection Serv ice (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility, under the Plant Protection Act (7 U.S.C. § 7701- 7772) to prevent the introduction and dissemina tion of plant pests into the U.S. The APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longer should be regulated . If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing

unrestricted introduction of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status in whole for the new biotechnology-derived, nu tritionally improved soybean product, MON 87705, any progeny deri ved from crosses between MON 87705 and conventional soybean, and any progeny de rived from crosses of MON 87705 with other biotechnology-derived soybean that has been granted nonregulated status under 7

CFR Part 340.

I.B. Rationale for the Development of Nutritionally Improved Soybean

MON 87705

Monsanto Company has developed biotec hnology-derived soybean MON 87705 with an improved fatty acid profile to enhance the suitability of soybean oil for food and industrial uses. The fatty acid (FA) leve ls in MON 87705 soybean oil are lower for saturated fats (6% vs. 15% FA) and higher for ol eic acid (76% vs. 23%). The increase in monounsaturated fatty acid (oleic) is acc ompanied by an overall decrease in polyunsaturated fatty acids ( 17% vs 60% FA). Conventi onal soybean oil typically contains 60-65% polyunsaturated fatty acids (P UFA’s), mostly in the form of linoleic acid. This high PUFA content makes soybean oil unsuitable for ma ny food applications since the high concentrations of PUFA’s in the oil are su sceptible to oxidation and degradation at high temperature.

To improve the stability of soybean oil, the polyunsaturated fatty aci ds in the oil can be decreased through a process called hydrogenation that redu ces the number of unstable double bonds found in fatty acids such as linol enic and linoleic, and converts them to saturated fats. Although hydrogenation produces oil with excellent thermal and oxidative stability, it also results in the pr oduction of signif icant levels of trans fatty acids in the

oil. Trans fatty acids contribute to cardiovascular risk by elevating LDL (bad cholesterol) and reducing HDL (good cholesterol) (K ris-Etherton, 1995; Hu et al., 1997).

Soybean oil oxidative stability can be improved because the stability of vegetable oils is drastically influenced by the proportion of monounsaturates to polyunsaturates. High oleic soybean oils are estimated to have improved oxidative stability up to 17.5 times greater than conventional soybean oil (Frankel, 2005). Oil from MON 87705 would similarly have an enhanced oxidative stab ility (approximately three-fold) relative toMonsanto Company 09-SY-201U Page 33 of 471 conventional soybean oil due to its increase in monounsaturated fatty acid and decrease in PUFA’s, without the need for hydrogena tion. Therefore, MON 87705 soybean oil can provide the food industry with options of enhanced stability for food formulation.

Numerous global health authoriti es recognize that diets high in total fat and saturated fat

are associated with increased risk of chr onic disease (FAO/WHO, 2002; Lichtenstein et al., 2006; IO, 2002; USHHS, 1988) , and health experts, in cluding the American Heart Association (Eckel et al., 2007), have reco gnized that an unintended consequence of a

shift to oils with no or lower trans fat levels, may be an increase in levels of saturated fats in foods. With a fatty acid profile lower in saturated fats than commodity soybean oil, and a reduced need for hydrogenation, MON 87705 soybean oil can help address this concern as MON 87705 soybean oi l contains less than 7% saturated fatty acids (palmitic + stearic acid). Based on U.S. Food and Drug Administration (F DA) guidance, a low

saturated fat food contains less than 1 g of fat per serving (www.fda.gov ) and the typical serving size for soybean oil is 14g (www.thumboilseed.com/soy-oil.htm ). As a result, to qualify as a low saturate soybean oil the maxi mum amount of satura ted fats allowed is 7% (0.98 g per serving). Therefore, unde r FDA guidance MON 87705 soybean oil can

be classified as a low saturate oil.

The fatty acid profile of MON 87705 soybean oil is also well suited for industrial applications. Soybean oils have very good lubricating properties, and are highly biodegradable compared to mine ral oils, but typically lack th e stability needed to meet industrial requirements. Hydrogenation of soybean oil is not ac ceptable for most

industrial uses because it leads to formation of saturated and trans fatty acids which can cause the oil to be solid at lower temperatur es, resulting in excessive wear and tear of machinery (Kinney, 1998). The fatty acid profile of MON 87705 provides an industrial oil with improved stability that could serve as a lubricant without needing hydrogenation. In addition, soybean oil with elevated oleic acid is an attractive sour ce for other industrial applications, such as petrochemical-derived plasticizers (Kinney, 1998). The higher oleic

acid and lower saturated fat le vels of MON 87705 also make it much more suitable for

use in biodiesel due to its greater stability, improved cold weather performance, and reduced nitrous oxides emissions (K nothe 2005; Bringe, 2005; Graef, 2009)

As with all new biotechnology-de rived traits, MON 87705 will al so be bred into soybean varieties with diverse genetic backgrounds. These varieties will include commercial

varieties with low linolenic acid levels whic h can further enhance the oxidative stability of the soybean oil. In addition, MON 87705 will be combined using traditional breeding

methods with other biotec hnology-derived traits, incl uding glyphosate tolerance (MON 89788), to deliver the best agronomic platform to farmers.

To summarize, MON 87705 was developed to improve soybean oil’s oxidative stability profile, without the need for hydr ogenation, and lower the saturate d fat content of the oil. Due to the compositional improvement in MON 87705 soybean oil, MON 87705 could expand the food market applicab ility of soybean oil, wit hout contributing further to known dietary health risks or sacrificing food flavor. Similarly, MON 87705 soybean oil could also serve as an improved source for industrial and biofuel products.Monsanto Company 09-SY-201U Page 34 of 471 I.C. Gene Suppression of FATB and FAD2 and Resulting Fatty Acid Composition

of the Seed

Suppression of FATB and FAD2 RNAs in so ybean decreases saturated fats (16:0 palmitic acid and 18:0 stearic acid), increase s oleic acid (18:1), and decreases linoleic acid (18:2).

The improved fatty acid prof ile in MON 87705 soybean oil is achieved through the use of endogenous soybean ( Glycine max L.) gene segments configured to suppress FATB and

FAD2 gene expression. MON 87705 contains FATB1-A and FAD2-1A gene segments under the control of a seed promoter, limiting oi l composition modification to this tissue. The assembled gene transcript has an inverted repeat th at produces double stranded RNA (dsRNA) that, via the RNA interference (RNAi) pathway, suppresses endogenous FATB

and FAD2 gene expression, thereby producing the desired fatty acid phenotype. Acylacyl carrier protein (ACP) thioesterases (re ferred to herein as FATB enzymes) are localized in plastids and hydrol yze saturated fatty acids from the ACP-fatty acid moiety. The suppression of FATB results in a decrease in the transport of the saturated fats out of the plastid, thus retaining their availability for desaturation to 18:1 oleic acid (see Figure I-1). Therefore, suppression of FATB decreases saturated fat content in the oil as well as increasing oleic acid (Kinney, 1996). Subsequently, this incr eased amount of oleic acid is either delivered to the oil body or e ndoplasmic reticulum for further desaturation (Kinney, 1996). Delta-12 desatu rases (referred to as FAD2 enzymes) desaturate 18:1 oleic acid to 18:2 linoleic acid. The suppr ession of FAD2 in soybean seed causes

reduced desaturation of oleic to linoleic acid thus contributing further to the increase in oleic while reducing linoleic acid content in the oil (Dyer and Mullen, 2005). Therefore, the overall result of the suppression of these two enzymes is a reduction in saturated 16:0 palmitic and 18:0 stearic fatty acids, an incr ease in monounsaturated 18:1 oleic acid, and lower levels of polyunsaturated 18:2 linolei c acid relative to commodity soybean.

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is mediated by dsRNA molecules. Doubl e stranded RNAs are commonly found in eukaryotes, including plants, for endogenous gene suppression and are composed of nucleic acids. Nucleic acids have a long hist ory of safe consumption and are considered GRAS by the U.S. Food and Drug Administra tion. There is no ev idence to suggest dietary consumption of RNA is associated wi th toxicity or allergenicity. Moreover, analysis of the DNA segments encoding this ds RNA showed that they lack the sequences required for translation initiation and protein synthesis. The production of a protein from the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several biotechnology-derived plant pr oducts previously deregulated by APHIS were developed using RNA-based suppression mechanisms, incl uding virus-resistant papaya and squash, high oleic soybean, FLAVR SAVR tomatoes, a nd plum trees resistant to Plum pox virus (FDA, 1994; USDA-APHIS, 1994; USDA-A PHIS, 1997; USDA-APHIS, 2006; USDAAPHIS, 2007). Based on this information, it is concluded that the inserted DNA and resulting dsRNA are safe and unl ikely to produce a protein or polypeptide. As a result, the RNA-based suppression technology used in MON 87705 poses no novel risks from a food, feed or environmental perspective.Monsanto Company 09-SY-201U Page 35 of 471 To examine suppression of the RNA levels of the endogenous FAD2-1A and FATB1-A genes, immature seed 2 at stage R5/6 were subjected to northern blot analyses and compared to a conventional control 3 that has a genetic background similar to the test substance. The details of the materials and methods are described in Appendix J.

To confirm the outcome of gene suppre ssion on the seed fatty acid profile of MON 87705, fatty acid analysis was pe rformed on MON 87705 compared to a conventional control. This analysis i ndicates the impact of the suppression of FATB and FAD2 genes on fatty acid profiles. The details of the full compositional analyses of MON 87705 are in Section VII, and the mate rials and methods for these analyses are described in Appendix E from Section VII.

Northern blot analysis of FAD2-1A RNA level in MON 87705 confirms suppression.

PolyA enriched RNA (PolyA+ RNA) isolated from approximately 50 μ g total RNA from immature seed was resolved on a formaldehyde /agarose gel, blotte d, and hybridized to the FAD2-1A probe (Figure I-2A). Approxi mately 200 pg of the FAD2-1A probe template was loaded on the gel to serve as a positive control, and the probe template produced a hybridization signal at approx imately 0.4 kb (Figure I-2A, lane 9). Approximately 50 pg of actin probe template wa s loaded on the gel to serve as a negative control and, as expected, there is no hybridizat ion signal produced (Figure I-2A, lane 10). The detection of the probe temp late hybridization c ontrol demonstrates that the probe is hybridizing only to the target DNA sequence.

PolyA+ RNA from each of four replicates of the conventional soybean control immature seeds (Figure I-2A, lanes 1, 3, 5, and 7) produced a strong hybrid ization signal at approximately 1.5 kb which is the expected size of the FAD2-1A transcript, whereas polyA+ RNA isolated from each of the four replicates of MON 87705 immature seeds (Figure I-2A, lanes 2, 4, 6, and 8) produced a very faint hybridization signal of approximately 1.5 kb at a greatly reduced level relative to th e conventional control RNA. There were faint, nonspecifi c hybridization signals observe d at approximately 1.7 kb, 3.2 kb, and 3.6 kb, which were detected in both the conventional soybean control and

MON 87705. These data show a reducti on in the levels of detectable FAD2-1A RNA in MON 87705 compared to conventional soybean.

In order to confirm that the reduced FAD2-1A RNA levels in MON 87705 was not due to a reduced RNA loading on the gel or poor RNA quality, the FAD2-1A hybridization signal was removed and the blot was re-hybridi zed with the actin probe (Figure I-2B). The approximately 50 pg of actin probe template that was loaded on the gel as a positive hybridization control showed a band at approximately 0.9 kb (Figure I-2B, lane 10). There is a faint band at approximately 1.8 kb, which most likely resulted from dimerization of the actin probe template. In lane 9, there is a band at approximately 0.4 kb, which likely results from the incomplete removal of the FAD2-1A probe template.

2 For studies presented throughout this pe tition referring to the use of MON 87705, conventional control, or reference material seed, the term “seed” refers to the harvested seed.

3 For studies presented throughout this petition comparing MON 87705 to the conv entional control, the conventional control was a nontransgenic parental soybean with genetics similar to MON 87705, but lacked the introduced traitsMonsanto Company 09-SY-201U Page 36 of 471 There is no hybridizatio n signal with the actin probe at approximately 0.9 kb, as expected from a negative control (Figure I-2B, lane 9). The detection of the actin probe template hybridization control de monstrates that the probe is hyb ridizing only to the target DNA

sequence.

PolyA+ RNA from conventional (Figure I-2B, lanes 1, 3, 5, and 7) and MON 87705 immature seeds (Figure I-2B, lanes 2, 4, 6, a nd 8) showed a strong hybridization signal at approximately 1.6 kb, which is expected for the actin transcript . The hybridization signals from conventional soybean and MON 87705 immature seeds have similar intensities, indicating that the RNA lo ading, RNA quality, and hybridization between conventional and MON 87705 are similar. Therefore, the difference in the FAD2-1A hybridization signals between conventional and MON 87705 reflects a large decrease in

FAD2-1A RNA level.

Northern blot analysis of FATB1-A RNA level in MON 87705 confirms suppression.

PolyA+ RNA isolated from approximately 100 μ g total RNA from immature seed was resolved on a formaldehyde/agarose gel, blo tted, and hybridized to the FATB1-A probe (Figure I-3A). Approximately 10 pg of the FA TB1-A probe template was loaded on the gel to serve as a positive control. As expected, the probe template produced a hybridization signal at approximately 0.4 kb (Fi gure I-3A, lane 9). Approximately 50 pg of the actin probe template was loaded on the gel to serve as a negative control and, as expected, there is no signal produced (Figur e I-3A, lane10). The detection of the FATB1-A probe template hybridization cont rol demonstrates that the probe is hybridizing only to the target DNA sequence.

PolyA+ RNA from four replicates of the immature seeds from conventional soybean (Figure I-3A, lanes 1, 3, 5, and 7) pr oduced a strong hybridization signal at approximately 1.8 kb, which is expected for the FATB1-A transcript, whereas polyA+ RNA isolated from four replicates of M ON 87705 immature seeds (Figure I-3A, lanes 2, 4, 6, and 8) also produced a hybridization signal of approxi mately 1.8 kb, but at a much reduced level. In addition, there was a hyb ridization signal at approximately 1.5 kb, which was not present in the conventional soybean control. The approximately 1.5 kb signal is likely a degraded FATB1-A transcript. These data i ndicate a reduction in the

RNA levels of the FATB1-A in MON 87705 compared to conventional soybean.

In order to confirm that the difference in the hybridization of the FATB1-A probe in

MON 87705 is not due to a reduced RNA lo ading on the gel or poor RNA quality, the FATB1-A hybridization signal was removed a nd the blot re-hybridized with the actin probe after stripping (Figure I-3B). The appr oximately 50 pg of actin probe template that was loaded on the gel as a positive contro l showed a band at approximately 0.9 kb (Figure I-3B, lane 10). The FATB1-A probe template did not produce any signal, as expected from a negative control (Figure I-3B, lane 9). The detection of the actin probe template control demonstrates that the pr obe is hybridizing only to the target DNA

sequence.

PolyA+ RNA from the conventional soybean control (Figure I-3B, lanes 1, 3, 5, and 7) and MON 87705 immature seed (Figure I-3B, lanes 2, 4, 6, and 8) showed a strong hybridization band at approximate ly 1.6 kb, which is expected fo r the actin transcript andMonsanto Company 09-SY-201U Page 37 of 471 the intensity of the bands are similar betw een the conventional control and MON 87705.

These data indicate that the RNA loadi ng, RNA quality, and hybridization between conventional soybean and MON 87705 are simi lar. Therefore the difference in the FATB1-A hybridization signals between conventional and MON 87705 reflects the

difference in FATB1-A RNA level.

Fatty Acid Composition of MON 87705shows predicted phenotype.

An assessment of the seed fatty acid profile co llected from five field sites in Chile during

2007/2008 demonstrates MON 87705 has the inte nded fatty acid changes. MON 87705 had a decrease in saturated fats (from 15.33% to 5.67% FA: 16:0 palmitic acid plus 18:0 stearic acid), an increase in oleic acid (f rom 22.81% to 76.47% FA), and a decrease in linoleic acid (from 52.86% to 10.10% FA). Statistically signif icant differences reflecting intended seed fatty acid change s were observed in the combined-site analysis (Table I-1) and at each individual site (Appendix E).

Conclusion

The suppression of FATB in soybean seed decreases bot h of the major saturated fatty acids (16:0 palmitic acid and 18:0 stearic acid), and suppression of FAD2 in soybean seeds increases oleic acid (18:1) and subs equently decreases linoleic acid (18:2) in soybean oil (Dyer and Mullen, 2005). M ON 87705 northern blot data confirms the suppression of endogenous FAD2-1A and FATB1-A RNAs. MON 87705 seed composition data demonstrates that suppressi on of these endogenous RNAs produces the intended alteration in fatty acid profile, which is a lower level of saturated fats (16:0 palmitic acid and 18:0 stearic acid), a higher level of oleic acid (18:1), and an associated lower level of linoleic acid (18:2).Monsanto Company 09-SY-201U Page 38 of 471 Figure I-1. Schematic of the Soybean Fatty Acid Biosynthetic Pathway indicates suppression of endogenous FATB and FAD2 in MON 87705 seedsMonsanto Company 09-SY-201U Page 39 of 471 Figure I-2. Northern Blot Analysis of FAD2-1A RNA Level in MON 87705

Panel A and Panel B are the same northern blot containing polyA+ RNA isolated from stage R5/6 immature seed tissue. Panel A is hybridized with the FAD2-1A probe. Panel B is hybridized with the actin probe after re moval of the FAD2-1A signal. Arrow heads indicate FAD2-1A hybridization signals and st ars indicate the actin hybridization signal. Lane designations are as follows:

Lane 1: Conventional contro l (PolyA+ RNA replicate 1) 2: MON 87705 (PolyA+ RNA replicate 1)

3: Conventional control (PolyA+ RNA replicate 2) 4: MON 87705 (PolyA+ RNA replicate 2)

5: Conventional control (PolyA+ RNA replicate 3) 6: MON 87705 (PolyA+ RNA replicate 3) 7: Conventional control (P olyA+ RNA replicate 4) 8: MON 87705 (PolyA+ RNA replicate 4) 9: FAD2-1A probe template (200 pg) 10: Actin probe template (50 pg)

Symbol denotes size of RNA, in base pairs, obtained from MW markers on ethidium stained gel.

4000

3000

2000

1500

1000

500

200 bp: 1 2 3 4 5 6 7 8 9 10 

### A B

* * 6000

4000

3000

2000

1500

1000

500

200 bp:

6000 1 2 3 4 5 6 7 8 9 10

4000

3000

2000

1500

1000

500

200 bp: 1 2 3 4 5 6 7 8 9 10 

### A B

* * 6000

4000

3000

2000

1500

1000

500

200 bp:

6000 1 2 3 4 5 6 7 8 9 10Monsanto Company 09-SY-201U Page 40 of 471 Figure I-3. Northern Blot Analysis of FATB1-A RNA Level in MON 87705

Panel A and Panel B is the same northern blot containing polyA+ RNA isolated from stage R5/6 immature seed tissue. Panel A is hybridized with the FATB1-A probe. Panel B is hybridized with the actin probe after removal of the FATB1-A signal. Arrow heads indicate FATB1-A hybridization signals and stars indicate th e actin hybridization signal. Lane designations are as follows:

Lane 1: Conventional c ontrol (PolyA+ RNA replicate 1) 2: MON 87705 (PolyA+ RNA replicate 1)

3: Conventional control (PolyA+ RNA replicate 2) 4: MON 87705 (PolyA+ RNA replicate 2)

5: Conventional control (PolyA+ RNA replicate 3) 6: MON 87705 (PolyA+ RNA replicate 3) 7: Conventional control (P olyA+ RNA replicate 4) 8: MON 87705 (PolyA+ RNA replicate 4) 9: FATB1-A probe template (10 pg) 10: Actin probe template (50 pg)

Symbol denotes size of RNA, in base pairs, obtained from MW markers on ethidium stained gel 6000

4000

3000

2000

1500

1000

500

200 bp: 1 2 3 4 5 6 7 8 9 10

6000

4000

3000

2000

1500

1000

500

200 bp: 1 2 3 4 5 6 7 8 9 10 

### A B

* *Monsanto Company 09-SY-201U Page 41 of 471 Table I-1. Summary of Product Concept Fa tty Acid Levels for Test (MON 87705)
vs. the Conventional Control (A3525) and Commercial Tolerance Interval

Component (Units) MON 87705

Mean [Range] A3525

Mean [Range] Commercial

Tolerance

Interval 1

Statistical Differences Observed in Combin ed-Site Analysis Seed Fatty Acid (% Total FA)

16:0 Palmitic 2.36 [2.25 - 2.44] 10.83 [10.51 – 11.08] [7.62, 12.55]

18:0 Stearic 3.31 [3.07 - 3.82] 4.50 [4.24 – 4.85] [2.87, 7.15]

18:1 Oleic 76.47 [73.13 - 79.17] 22.81 [21.41 – 25.08] [18.40, 30.22]

18:2 Linoleic 10.10 [7.85 - 12.42] 52.86 [51.68 – 53.89] [47.75, 56.46]

1 With 95% confidence, interval contains 99% of the values expresse d in the population of commercial soybean varieties

I.D. Submissions to Other Regulatory Agencies

Under the Coordinated Framework for Regul ation of Biotechnology, the responsibility for regulatory oversight of biotechnology-derived crops that do not include plantincorporated protectants falls on two federal agencies: FDA and USDA. Deregulation of MON 87705 by USDA constitutes only one component of the overall regulatory oversight and review of th is product. As a practic al matter, MON 87705 cannot be

released and marketed until FDA and US DA have completed their reviews and assessments under their resp ective jurisdictions.

Submission to FDA

MON 87705 falls within the scope of the 1992 U.S. Food and Drug Administration’s (FDA) policy statement concerning regulat ion of products derived from new plant varieties, including those developed thr ough biotechnology (FDA, 1992). In compliance with this policy, Monsanto w ill initiate a consultation with the FDA on the food and feed safety and nutritional assessment summary for MON 87705.

Submissions to Foreign Government Agencies

To support commercial introduction of MON 87705 in the U.S., re gulatory submissions

will be made to countries that import significant quantities of soybean or its processed

fractions from the U.S. and have establis hed regulatory approval processes in place.

These will include submissions to a number of foreign government regulatory authorities,Monsanto Company 09-SY-201U Page 42 of 471 including: Ministry of Agri culture, People’s Republic of Ch ina; Japan’s Ministry of Agriculture, Forestry, and Fisheries (MAFF) and the Ministry of Health, Labor, and Welfare (MHLW); the Canadian Food Inspectio n Agency (CFIA) and Health Canada; the Intersectoral Commission for Biosafety of Genetically Modified Organisms (CIBIOGEM), Mexico; the European Food Safe ty Authority (EFSA); and the regulatory authorities in other soybean importing countries with functioning regul atory systems. As appropriate, notifications of importation will be made to importing countries that do not have a formal approval process.Monsanto Company 09-SY-201U Page 43 of 471 II. THE SOYBEAN FAMILY

This section summarizes the taxonomy, biol ogy, and use of soybean based on: 1) the

consensus document for Glycine max (L.) Merr. prepared by the Organization for Economic Co-operation and Developmen t (OECD, 2000; OECD, 2001), and 2) a summary prepared by USDA-APHIS (2006) and a biology document published by Canadian Food Inspection Agency-Pla nt Biosafety Office (CFIA, 1996).

II.A. Soybean as a Crop

Soybean is the most prevalen tly grown oilseed in the worl d, with approximately 222.1 million metric tons (MMT) of harvested s eed produced in 2007, which represented 56% of world oilseed seed production that year (ASA, 2008; Soyatech, 2008). Soybean is grown as a commercial crop in over 35 countri es. The major producers of soybean are the U.S., Brazil, Argentina, China, and Indi a, which accounted for approximately 91% of the global soybean production in 2007 (Soyat ech, 2008); also see Table II 1. Approximately one-third of the 2007 world so ybean production was produced in the U.S. (Soyatech, 2008). The soybean produced in China and India are primarily for domestic use, while a significant portion of soybean pr oduced in U.S., Brazil, and Argentina is traded globally in the form of soybean harv ested seed, soybean meal, or soybean oil. Globally, the U.S. was the largest soybean s eed export country, while Argentina led the soybean meal and soybean oil export ma rkets in 2007 (ASA, 2008; Soyatech, 2008).

Table II-1. World Soybean Production in 2007/2008

Country Production (million metric tons)

U.S. 71.4

Brazil 61.0 Argentina 47.0

China 15.6

Other 8.9

India 7.9
Paraguay 6.2

Canada 3.1

EU 1.0

Source: Soya and Oilseed Bluebook (Soyatech, 2008).

Approximately 50% of the world soybean s eed supply was crushe d to produce soybean meal and oil in 2007 (ASA, 2008; Soyatech, 20 08), and the majority was used to supply the feed industry for livestoc k use or the food industry fo r edible vegetable oil and soybean protein isolates . Another 34% of the world soyb ean seed supply was traded to other geographies, with China, EU, Japan, and Mexico being the top soybean seed import geographies (ASA, 2008). The remainder of the soybean seed produced was used as certified seed, feed, or stocks.

Soybean is used in various food products, including tofu, so ybean sauce, soymilk, energy bars, and meat products. A major food use for soybean is purified oil, for use in margarines, shortenings, cooking, and salad oils. Soybean oil generally has a smallerMonsanto Company 09-SY-201U Page 44 of 471 contribution to soybean’s overall value comp ared to soybean meal because the oil constitutes just 18 to 19% of the soybean's weight. Nonetheless, soybean oil accounted for approximately 30% of all the vegetable oils consumed globally, and was the second largest source of vegetable oil worldwide, s lightly behind palm oil at approximately 32% share (Soyatech, 2008).

Soybean meal is used as a s upplement in feed rati ons for livestock. S oybean meal is the most valuable component obtained from processing the soybean, accounting for roughly 50-75% of its overall value. By far, soybean meal is the world's mo st important protein feed, accounting for nearly 69% of world prot ein meal supplies (ASA, 2008). Industrial uses of soybean range from a carbon/nitroge n source in the producti on of yeasts via fermentation to the manufacture of soaps, in ks, paints, disinfectan ts, and biodiesel. Industrial uses of soybean have been su mmarized by Cahoon (2003) and the American Soybean Association (ASA, 2008).

Global soybean plantings reached 90.8 milli on hectares in 2007/08, an 8.9% increase over the previous four years, with an av erage of 82.3 million hectares planted from 2002/03 to 2007/08 (Soyatech, 2008). Soybean production has realized, on average, a 6.2% annual growth between 1995/96 to 20 06/07. Increased planting flexibility, increased yield from narrow-row seeding pr actices, a higher rate of corn-soybean rotations, and low production co sts favored expansion of soybean areas in the mid-1990s, and the expanded areas tended to be concentrat ed where soybean yields were highest.

II.B. History of Soybean

Domestication of soybean is thought to have taken pla ce in China during the Shang dynasty (approximately 1500 to 1027 B.C.) or earlier (Hymowitz, 1970). However, historical and geographical evidence only could be traced back to the Zhou dynasty (1027 to 221 B.C.) where the soybean was utilized as a domesticated crop in the northeastern part of China. By the first century A. D., the soybean probably reached Central and Southern China as well as pe ninsular Korea. Th e movement of soybean germplasms was probably associated with the development and consolidation of territories and the degeneration of Chinese dynastie s (Ho, 1969; Hymowitz, 1970).

From the first century A.D. to approximat ely the 15th and 16th centuries, soybean was

introduced into several count ries, with land races eventu ally developing in Japan, Indonesia, Philippines, Vietnam, Thailand, Malaysia, Myanmar, Nepal, and Northern India. The movement of soybean throughout this period was due to the establishment of sea and land trade routes, the migration of certain tribes from China, and the rapid acceptance of seeds as a staple food by ot her cultures (Hymowitz and Newell, 1981; Hymowitz et al., 1990).

Starting in the late 16th century and thr oughout the 17th century, soybean was used by the Europeans, and in the 17th century, soyb ean sauce was a common item of trade from

the East to the West.

Soybean was introduced into North America in the 18th century. In 1851, soybean was

introduced in Illinois and subse quently throughout the Corn Be lt. In 1853, soybean seed were deposited at the New York State Ag ricultural Society, the Massachusetts Horticultural Society, and the Commissioner of Patents. The two societies and theMonsanto Company 09-SY-201U Page 45 of 471 Commissioner of Patents sent soybean seed to dozens of growers throughout the U.S. Soybean has been cultivated extensivel y and improved through conventional breeding following its introduction in the U.S. and subsequently has become a key source of

nutrients for food and feed use in th e U.S. (Hymowitz and Singh, 1987).

II.C. Taxonomy and Phylogenetics of Soybean

Cultivated soybean, Glycine max (L.) Merr., is a diploidize d tetraploid (2n=40), which belongs to the family Leguminosae, the subf amily Papilionoideae, the tribe Phaseoleae, the genus Glycine Willd., and the subgenus Soja (Moench) F.J. Herm.

Family: Leguminosae

Subfamily: Papilionoideae

Tribe: Phaseoleae

Genus: Glycine

Subgenus: Soja (Moench) F.J. Herm.

Species: max

The genus Glycine Willd. is of Asian and Australian origin and is divided into two subgenera, Glycine and Soja (Moench) F.J. Herm. The subgenus Glycine consists of 22 wild perennial species, which are indige nous to Australia, West, Central and South Pacific Islands, China, Russia, Japan, Indonesia, Korea, Papua New Guinea, the Philippines, and Taiwan (Hymowitz, 2004). The subgenus Soja includes the cultivated soybean, G. max (L.) Merr. and its wild annual relatives from Asia, G. soja Sieb. and Zucc. The list of species in the genus Glycine Willd. is presented in Table II-2.Monsanto Company 09-SY-201U Page 46 of 471 Table II-2. List of Species in the Genus Glycine Willd., 2n Chromosome Number,
Genome Symbol, and Distribution

Genus 2n Genome 1 Distribution

Subgenus Glycine

1. G. albicans Tind. & Craven 40 I1 Australia

2. G. aphyonota B. Pfeil 40

-- 2 Australia

3. G. arenaria Tind. 40 HH Australia

4. G. argyrea Tind. 40 A2A2 Australia

5. G. canescens F.J. Herm. 40 AA Australia

6. G. clandestina Wendl. 40 A1A1 Australia

7. G. curvata Tind. 40 C1C1 Australia

8. G. cyrtoloba Tind. 40 CC Australia

9. G. dolichocarpa Tateishi and Ohashi 80

-- (Taiwan)

10. G. falcate Benth. 40 FF Australia

11. G. hirticaulis Tind. & Craven 40 H1H1 Australia

80

-- Australia

12. G. lactovirens Tind. & Craven. 40 I1I1 Australia

13. G. latifolia (Benth.) Newell & Hymowitz 40 B1B1 Australia

14. G. latrobeana (meissn.) Benth. 40 A3A3 Australia

15. G. microphylla (Benth.) Tind. 40 BB Australia

16. G. peratosa B. Pfeil & Tind. 40

-- Australia

17. G. pindanica Tind. & Craven 40 H3H2 Australia

18. G. pullenii B. Pfeil, Tind. & Craven 40

-- Australia

19. G. rubiginosa Tind. & B. Pfeil 40

-- Australia

20. G. stenophita B. Pfeil & Tind. 40 B3B3 Australia

21. G. tabacina (Labill.) Benth. 40 B2B2 Australia

80 Complex 3 Australia, West Central and

South Pacific Islands

22. G. tomentella Hayata 38 EE Australia

40 DD Australia, Papua New Guinea

78 Complex 4 Australia, Papua New Guinea

80 Complex 5 Australia, Papua New Guinea, Indonesia, Philippines, Taiwan Subgenus Soja (Moench) F.J. Herm.

23. G. soja Sieb. & Zucc. 40 GG China, Russia, Taiwan, Japan, Korea (Wild Soybean)

24. G. max (L.) Merr. 40 GG Cultigen (Soybean)

1 Genomically similar species carry the same letter symbols.

2 Genome designation has not b een assigned to the species.

3 Allopolyploids (A and B genomes) and segmental allopolyploids (B genomes).

4 Allopolyploids (D and E, A and E, or any other unknown combination).

5 Allopolyploids (A and D genomes, or any other unknown combination). Note: Table is adapte d from Hymowitz (2004).

Glycine soja grows wild in China, Ja pan, Korea, the Russian Fa r East, and Taiwan, and is commonly found in fields, hedgerows, roadsides, and riverbanks (Lu, 2004). The plant isMonsanto Company 09-SY-201U Page 47 of 471 an annual, slender in build wi th narrow trifoliolate leaves . The purple or very rarely

white flowers are inserted on sh ort, slender racemes. The pods are short and tawny with hirsute pubescence, producing oval- oblong seeds (Hermann, 1962).

Glycine max (L.) Merr., the cultivated soybean, is an annual that generally exhibits an erect, sparsely branched, bush-type growth hab it with trifoliolate leaves. The leaflets are broadly ovate, and the purple, pink, or white flowers are borne on short axillary racemes or reduced peduncles. The pods are either stra ight or slightly curved, and one to three ovoid to subspherical seeds are produced per pod.

A third and unoffici al species named G. gracilis is also described within the context of

the Soja subgenus in addition to G. soja and G. max . The G. gracilis is known only from Northeast China, is interm ediate in morphology between G. max and G. soja , and is

sometimes considered a variant of G. max . The three species in the Soja subgenus can cross-pollinate, and the hybrid seed can germinate normally and subsequently produce fertile pollen and seed (Singh and Hy mowitz, 1989). The taxonomic position of G. gracilis has been an area of debate, and neith er ILDIS (International Legume Database and Information Service) nor USDA-GRIN (USDA Germplasm Reso urces Information Network) recognizes G. gracilis as a distinct species. The wild and weedy relatives ( G.
soja and G. gracilis ) of soybean do not occur in the U.S ., and, therefore, are not likely to contribute to the poten tial for outcrossing (USDA-APHIS, 2006).

II.D. The Genetics of Soybean

Glycine is the only genus in the tribe Phaseoleae where species have diploid chromosome numbers of 40 and 80, but not 20 (Lackey, 1981). The unique chromosome number of Glycine is probably derive d from diploid ance stors with base num ber of 11. The ancestral species have under gone aneuploid reduction (loss of a specific chromosome), which is prevalent throughout the Papilionoide ae, to a base number of 10 chromosomes (Lackey, 1981). Te traploidization (2 n = 2 x = 40) through autopolyploidy or allopolyploidy of the progenitor species occurred either prior to or after dissemination from the ancestral region. The path of mi gration from a common progenitor is assumed by (Singh et al., 2001) as : wild perennial (2 n = 4 x = 40, unknown or extinct) to wild annual (2 n = 4 x = 40; G. soja ) to soybean (2 n = 4 x = 40; G. max ). Soybean should be regarded as a stable tetraploid with diploidized genom e (Gurley, 1979; Lee and Verma, 1984; Skorupska, 1989).

II.E. Pollination of Cultivated Soybean

Soybean is a self-pollinated species, propagated by seed (OECD, 2000). The papilionaceous flower consists of a tubular caly x of five sepals, a co rolla of five petals, one pistil, and nine fused stamens with a sing le separate posterior stamen. The stamens form a ring at the base of the stigma and elongate one day before pollination, at which time the elevated anthers form a ring ar ound the stigma (OECD, 2000). The soybean flower stigma is receptive to pollen approxi mately 24 hours before anthesis and remains receptive for 48 hours after anthesis. The anther s mature in the bud and directly pollinate the stigma of the same flower. As a result , soybean is considered to be a highly selfpollinated species, with cross-pollination to adjacent plants of other soybean varietiesMonsanto Company 09-SY-201U Page 48 of 471 occurring at very low frequency (0 to 6.3% ) in adjacent plants (Caviness, 1966; Ray et al., 2003; Yoshimura et al., 2006). Pollination typically takes place on the day the flower opens. The pollen naturally comes in contact with the stigma during the process of anthesis. Anthesis normally occurs in late morning, depending on the environmental conditions. The pollen usually remains viable for two to four hours, and no viable pollen can be detected by la te afternoon. Natural or artificial cross-pollination can only take place during the short time when the pollen is viable.

II.F. Cultivated Soybean as a Volunteer

Cultivated soybean plants are annuals, and they reproduce solely by means of seeds. Volunteer soybean in rotationa l crops is typically not a c oncern in most environments where soybean is cultivated (CFIA, 1996; OECD, 2000). Soybean seed rarely exhibit any dormancy characteristics, and seed rema ining in the field after harvest likely will readily imbibe water (Lersten and Carlson, 2004), germinate, and will be killed by frost

or field cultivation. If they did become established, volunteer plants would not compete well with the succeeding crop, and could be controlled readily by either mechanical or chemical means (OECD, 2000).

II.G. Characteristics of the Recipient Plant

The soybean variety used as the recipien t for the DNA insertion to create MON 87705 was A3525, a nontransgenic conventional variet y developed by Asgrow Seed Company. A3525 is a mid-maturity Group II I soybean variety with very high yield potenti al. It has superior yields relative to lines of si milar maturity and has excellent agronomic characteristics (Steffen, 2004).

II.H. Soybean as a Test System in Product Safety Assessment

In developing the data to support the safe ty assessment of M ON 87705, A3525 was used as the nontransgenic conven tional soybean comparator. In general, the genetic background of MON 87705 was matched with that of the control so the effect of the genetic insertion and the pres ence of the CP4 EPSPS protein could be assessed in an

unbiased manner. Since MON 87705 was deri ved from the A3525 conventional variety, it was deemed appropriate to use the nont ransformed A3525 as the control variety because its use would minimize the poten tial bias in subsequent comparative assessments. In addition, commercial conventional and Roundup Ready soybean (40-3- 2) varieties were used as reference material s to establish ranges of responses or values representative of commercial soybean variet ies. The reference varieties used at each

location were selected based on their ava ilability and agronomic fit (Appendix F and Table F-1).Monsanto Company 09-SY-201U Page 49 of 471 III. DESCRIPTION OF THE TRANSFORMATION SYSTEM

MON 87705 was developed through Agrobacterium -mediated transformation of soybean meristem tissue using the doubl e-border, binary vector PV-GMPQ/HT4404 (Section IV, Figure IV-1). The vector, PV -GMPQ/HT4404, contains both the left and right border sequences flanking the transfer DNA (T-DNA) to facilitate transformation.

This vector, PV-GMPQ/HT4404, is approxima tely 13.1 kb and contains two T-DNAs, each delineated by left and ri ght border regions. The fi rst T-DNA, designated T-DNA I,

contains a cp4 epsps expression cassette and a partial suppression cassette. The cp4

epsps expression cassette is under the regulation of FMV/Tsf1 chimeric promoter and E9 polyadenylation sequence. Th e partial suppression cassett e in T-DNA I contains the sense segments of the FAD2-1A intron and FATB1-A 5' UTR, including the chloroplast targeting sequence, that are unde r the regulation of the seed 7S α ' promoter. The second T-DNA, designated T-DNA II, cont ains a partial suppression casse tte that consists of the antisense segments of FAD2-1A intron and FATB1-A 5 ′ UTR, which is flanked by the H6 untranslated sequence. During plant transfor mation, the two T-DNAs co-integrated at one locus in the soybean genome, cr eating a DNA insert that contains a cp4 epsps cassette and a single FAD2-1A and FATB1-A suppression cassette.

The Agrobacterium -mediated soybean transformati on to produce MON 87705 was based on the method described by Martinell et al . (2002), which allows the generation of transformed plants without util ization of callus. Briefly, meristem tissues were excised from the embryos of germinated A 3525 seed. After co-culturing with Agrobacterium carrying the vector, the meristems were placed on selection medium containing glyphosate to inhibit the growth of untransfor med plant cells, as well as carbenicillin and

Claforan to inhibit excess Agrobacterium . The meristems were then placed in media conducive to shoot and root development, and only rooted plants with normal phenotypic

characteristics were selected and transferred to soil for growth and further assessment.

The R 0 plants generated through this transformation were self-pollinated, and the subsequent R 1 plants were screened for the zygos ity of inserted gene. Only the R 1 plants that were homozygous for the insertion, as determined by quantitative PCR, and produced seeds with the desired fatty acid co mposition were advanced for development. Their progeny were subjected to furthe r phenotypic assessme nts. MON 87705 was selected as the lead event based on superi or phenotypic characteris tics, agronomics, and molecular profile. Regulat ory studies on MON 87705 were initiated to further characterize the genetic insertion and the ex pressed protein, and to establish the food, feed, and environmental safety relative to conventional soybean . The major steps involving the development of MON 87705 are depicted in Figure III-1.Monsanto Company 09-SY-201U Page 50 of 471 Figure III-1. Schematic of the Development of MON 87705Monsanto Company 09-SY-201U Page 51 of 471 IV. GENETIC ELEMENTS

This section describes the vector, the donor ge nes and the regulatory elements used in the development of MON 87705 and the deduced amino acid sequence of the CP4 EPSPS protein produced in MON 87705. In this section, T-DNA refers to DNA that is transferred to the plant during transformation. An expression cassette is comprised of sequences to be transcribed a nd the regulatory elements nece ssary for the expression of those sequences. The suppression cassette refers to the sequences and regulatory elements necessary for the suppression of the endogenous FAD2 and FATB RNA transcripts.

IV.A. Vector GMPQ/HT4404

The PV-GMPQ/HT4404 vector used for th e transformation of soybean to produce MON 87705 is shown in Figure IV-1 and its el ements described in Table IV-1. This vector is approximately 13.1 kb and contai ns two T-DNAs, each delineated by left and right border regions. T-DNA I contains a cp4 epsps expression cassette and a partial suppression cassette. The cp4 epsps expression cassette is under the regulation of FMV/Tsf1 chimeric promoter and E9 polyadenylation sequence. The partial suppression cassette in T-DNA I contains the sense segments of the FAD2-1A intron and FATB1-A 5' UTR, including the chloroplast targeting sequence, which ar e under the regulation of the

seed 7S α ' promoter. T-DNA II contains a partial suppression cassette, which consists of the antisense segment of FAD2-1A intron and FATB1-A 5 ′ UTR that is flanked by the H6 untranslated sequence. During plant transformation, a porti on of the plants that were generated contained the two T- DNAs co-integrated at one lo cus in the soybean genome creating a DNA insert that contains a cp4 epsps cassette and a single FAD2-1A and

FATB1-A suppression cassette.

The vector backbone region outsi de of the T-DNA contains two origins of replication for maintenance of plasmid in bacteria ( OR-oriV

, OR-ori-PBR322 ), a bacterial selectable marker gene ( aadA ), and a coding sequence for repr essor of primer protein for maintenance of plasmid copy number in E. coli ( rop ). A description of the genetic elements and their prefixes (e.g., P-, L-, I-, TS-, OR-, B-, CS-, and T-) in PV-GMPQ/HT4404 is provided in Table IV-1.

IV.B. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA I)

The cp4 epsps gene expression cassette is present in MON 87705. The cp4 epsps coding sequence encodes a 47.6 kDa CP4 EPSPS protein consisting of a sing le polypeptide of 455 amino acids (Padgette et al., 1996b). The cp4 epsps gene expression cassette was used as a selectable marker during the transformation to produce MON 87705. The CP4 EPSPS protein is similar and functionally id entical to endogenous plant EPSPS enzymes, but has a much reduced affinity for gl yphosate, the active in gredient in Roundup herbicides, relative to endogenous plant E PSPS (Padgette et al., 1996a). The CP4 EPSPS protein confers resistance to glyphosate and has been used safely and successfully in many Roundup Ready crops, such as canola, co rn, cotton, soybean, and sugar beet. The deduced CP4 EPSPS protein full-length amino acid sequence is shown in Figure IV-3.Monsanto Company 09-SY-201U Page 52 of 471 IV.C. The FAD2-1A and FATB1-A segments Sequence (T-DNA I and II)

MON 87705 contains a partial sequence of the soybean ( Glycine max ) FAD2-1A gene and FATB1-A gene. The FAD2-1A and FATB1-A gene segments are comprised of ~ 0.6 kb of sequence from the FAD2-1A intron and the FATB1-A 5 ′ UTR, and form the MON 87705 suppression cassette. This suppression cassette e xpresses an inverted repeat

of the FAD2-1A and FATB1-A gene segments. The assembled gene transcript has an inverted repeat that produces double st randed RNA (dsRNA) that, via the RNA interference (RNAi) pathwa y, suppresses endogenous FATB and FAD2 RNA, and, ultimately, an improved fatty acid composition in the seed.

IV.D. Regulatory Sequences

The cp4 epsps coding sequence that is located in T- DNA I, is under the regulation of the FMV/Tsf1 promoter, the Tsf1 leader and intron, the CTP2 targeting sequence and the E9 3' untranslated sequence. The FMV/Tsf1 is a chimeric promoter consisting of enhancer sequences from the promoter of the Figwort Mosaic virus 35S RNA (R ichins et al., 1987) combined with the promoter from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 alpha (Axelos et al., 1989). The Tsf1 leader is the 5’ untranslated region from the Arabidopsis thaliana Tsf1 gene (Axelos et al, 1989) that encodes the elongation factor EF-1 alpha. The CTP2 targ eting sequence is the sequence encoding the transit peptide from the ShkG gene of Arabidopsis thaliana (Klee et al., 1987) and is present to direct the CP4 EPSPS pr otein to the chloroplast. The E9 3 ′ untranslated region is the 3' untranslated sequence from the RbcS2 gene of Pisum sativum (Coruzzi et al., 1984) and is present to dire ct polyadenylation of the cp4 epsps transcript.

Also located in T-DNA I is a partial suppression cassette with a portion of the FAD2-1A

intron and a portion of the FATB1-A 5 ' UTR, including the plasti d targeting sequence under the regulation of the Glycine max 7S α ' seed storage gene promoter (designated Sphas 1) which drives expression in im mature seeds (Doyle et al., 1986).

T-DNA II contains another partial suppression cassette with the FAD2-1A and FATB1-A gene segments followed by the H6 3' untranslated sequence. The H6 3' untranslated region is from the Gossypium barbadense cotton fiber gene and is present to terminate transcription (John a nd Keller, 1995).

IV.E. T-DNA Borders

Plasmid PV-GMPQ/HT4404 contains right border and left bor der regions (Figure IV-1 and Table IV-1) that were derived from Agrobacterium tumefaciens plasmids (Barker et al., 1983; Zambryski et al., 1982). The border regions each c ontain a 24-25 bp nick site that is the site of DNA exchange during tran sformation. The border regions delineate the

T-DNA and are involved in their efficient transfer into th e soybean genome. Because PV-GMPQ/HT4404 is a two T-DNA vector, it contains two ri ght border regions and two left border regions, where one set flank T- DNA I and the other set flank T-DNA II (see description above).Monsanto Company 09-SY-201U Page 53 of 471 IV.F. Genetic Elements Outside of the T-DNA Borders

Genetic elements that exist outside of the T- DNA borders are those that are essential for

the maintenance and selection of the vector PV-GMPQ/HT4404 in bacteria. The origin of replication OR-ori V is required for the maintenance of the plasmid in Agrobacterium (Stalker et al., 1981) and is derived from th e broad host plasmid RK2. The origin of replication OR- ori.pBR322 is required for the maintenance of the plasmid in E. coli and is derived from the plasmid pBR322 (Sutcliffe, 1979). CS- rop is the coding sequence of the repressor of primer (ROP) protein and is necessary for the maintenance of plasmid copy number in E. coli (Giza and Huang, 1989). The selectable marker aadA is a bacterial promoter and coding sequence for an enzyme from trans poson Tn7 that confers spectinomycin and streptomycin re sistance (Fling et al., 1985) in E. coli and Agrobacterium during molecular cloning. Because these elements are outside the border regions, they are not expected to be transferred into the so ybean genome. The absence of the backbone sequence in MON 87705 has been confirmed by Southern blot analyses (see Section V.B.).Monsanto Company 09-SY-201U Page 54 of 471 Probe DNA Probe Start Position Stop Position Total Length (~kb)

1 T-DNA I Probe 1A 7657 9406 1.8

2 T-DNA I Probe 2B 9270 10042 0.8

3 T-DNA I Probe 3C 9943 11325 1.4

4 T-DNA I Probe 4D 11151 160 2.1

5 T-DNA I Probe 5E 13080 973 1.0

6 T-DNA II Probe 1A 5570 6693 1.1

Figure IV-1. Circular Map of Plasmid PV-GMPQ/HT4404 showing probes 1-6 Plasmid PV-GMPQ/HT4404 containing the T-DNAs used in Agrobacterium -mediated transformation to produce MON 87705. Genetic el ements (depicted in the exterior of the map) and restriction sites for enzymes used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The T-DNA I and T-DNA II probes used in the Southern analyses (labeled 1-6 within the interior of the map) are detailed in the accompanying table

. PV-GMPQ/HT4404

13088 bp Nco I 9850 Spe I 12631 Xho I 13088

B-Right Border

aadA

OR- ori.pBR322

CS- rop

B-Left Border

T- H6 B-Right Border OR- ori V B-Left Border P- FMV/Tsf1

L- Tsf1 I- Tsf1 TS- CTP2 CS- cp4 epsps T- E9 P- 7S α '

FAD2-1A p

FATB1A p FATB1A p FAD2-1A p

1 2 3 4 5

6

T-DNA II T-DNA IMonsanto Company 09-SY-201U Page 55 of 471 Probe DNA Probe Start Position Stop Position Total Length (~kb)

7 Backbone Probe 1 974 2280 1.3

8 Backbone Probe 2 2140 4080 1.9

9 Backbone Probe 3 3631 5127 1.5

10 Backbone Probe 4 7025 7656 0.6

Figure IV-2 Plasmid Map of Vector PV-GMPQ/HT4404 Showing Probes 7-10 Plasmid PV-GMPQ/HT4404 containing the T-DNAs used in Agrobacterium -mediated transformation to produce MON 87705. Genetic el ements (depicted in the exterior of the map) and restriction sites for enzymes used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of th e map. The backbone probes used in the Southern analyses (labeled 7-10 within the interior of the map) are detailed in the accompanying table. PV-GMPQ/HT4404

13088 bp Nco I 9850 Spe I 12631 Xho I 13088

B-Right Border

aadA

OR- ori.pBR322

CS- rop

B-Left Border

T- H6 B-Right Border OR- ori V B-Left Border P- FMV/Tsf1

L- Tsf1 I- Tsf1 TS- CTP2 CS- cp4 epsps T- E9 P- 7S α '

FAD2-1A p FATB1A p FATB1A p FAD2-1A p

T-DNA II T-DNA I

7

8

9

10Monsanto Company 09-SY-201U Page 56 of 471 Table IV-1. Summary of Genetic Elemen ts in Plasmid Vector PV- GMPQ/HT4404

Genetic Element Location in

Plasmid Function (Reference)

T-DNA I

B 1 -Left Border 7657 – 8098 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al., 1983) Intervening Sequence 8099 – 8134 Sequence used in DNA cloning

P 2

- FMV/Tsf1 8135 – 9174 Chimeric promoter consisting of enhancer sequences from the promoter of the Figwort Mosaic virus 35S RNA (Richins et al., 1987) combined with the promoter from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1alpha (Axelos et al., 1989)

L 3

- Tsf1 9175 – 9220 5' untranslated leader (exon 1) from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 al pha (Axelos et al., 1989)

I 4

- Tsf1 9221 – 9842 Intron with flanking exon sequence from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1alpha (Axelos et al., 1989)
Intervening Sequence 9843 – 9851 Sequence used in DNA cloning

TS 5

- CTP2 9852 – 10079 Targeting sequence from the ShkG gene encoding the transit peptide region of Arabidopsis thaliana EPSPS (Klee et al., 1987) th at directs transport of the CP4 EPSPS protein to the chloroplast

CS 6

- cp4 epsps 10080 –11447 Codon modified coding sequence of the aroA gene from the Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein (B arry et al., 1997; Padgette et al., 1996a)
Intervening Sequence 11448 – 11505 Sequence used in DNA cloning

T 7

- E9 11506 – 12148 3 ′ untranslated region of the pea RbcS2 gene which functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984)

Intervening Sequence 12149 – 12236 Sequence used in DNA cloning

P- 7S α ′ 12237 – 13077 Promoter and leader from the Sphas1 gene of Glycine max encoding beta-conglycinin storage protein (alpha'-bcsp) (D oyle et al., 1986) that directs transcription in seed

B 1 – Border; P 2 – Promoter; L 3 – Leader; I 4 – Intron; TS 5 – Targeting Sequence; CS 6 – Coding Sequence;

T 7 – 3' untranslated transcriptional termination sequence and polyadenylation signal sequences;Monsanto Company 09-SY-201U Page 57 of 471 Table IV-1 (cont.). Summary of Gene tic Elements in Plasmid Vector PVGMPQ/HT4404

Intervening Sequence 13078 – 11 Sequence used in DNA cloning

FAD2-1A p 12 – 277 Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 desaturase (Fillatti et al., 2003) which forms part of the suppression cassette

FATB1-A p 278 – 578 Partial sequence from the 5' untranslated region and the plastid targeting sequence from Glycine

max FATB1-A gene that encodes the palmitoyl acyl carrier protein thioesterase (Fillatti et al., 2003) which forms part of the suppression cassette
Intervening Sequence 579 – 616 Sequence used in DNA cloning

B-Right Border 617 – 973 DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA (Zambryski et al., 1982)

Vector Backbone
Intervening Sequence 974 – 1109 Sequence used in DNA cloning

aadA 1110 – 1998 Promoter, coding sequence, and 3' UTR for anaminoglycoside-modifying enzyme, 3''(9)-Onucleotidyltransferase from the transposon Tn7 (Fling et al., 1985) that confers spectinomycin and streptomycin resistance
Intervening Sequence 1999 – 2528 Sequence used in DNA cloning

OR 8

- ori-pBR322 2529 – 3117 Origin of replica tion from pBR322 for maintenance of plasmid in E. coli (Sutcliffe, 1979)

Intervening Sequence 3118 – 3544 Sequence used in DNA cloning

CS- rop 3545 – 3736 Coding sequence for repressor of primer protein from the ColE1 plasmid for maintenance of plasmid copy number in E. coli (Giza and Huang, 1989)
Intervening Sequence 3737 – 5127 Sequence used in DNA cloning

T-DNA II

B-Left Border 5128 – 5569 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al., 1983)

P

– Partial sequence; OR 8 – Origin of Replication.Monsanto Company 09-SY-201U Page 58 of 471 Table IV-1 (cont.). Summary of Gene tic Elements in Plasmid Vector PVGMPQ/HT4404

Intervening Sequence 5570 – 5667 Sequence used in DNA cloning

T- H6 5668 – 6103 3' UTR sequence of the H6 gene from Gossypium

barbadense encoding a fiber pr otein involved in secondary cell wall assembly (John and Keller, 1995)
Intervening Sequence 6104 – 6115 Sequence used in DNA cloning

FAD2-1A p 6116 – 6381 Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes th e delta-12 desaturase (Fillatti et al., 2003) which forms part of the suppression cassette

FATB1-A p 6382 – 6682 Partial sequence from the 5' untranslated region and the plastid targeting sequence from Glycine max

FATB1-A gene that encode s the palmitoyl acyl carrier protein thioesterase (Fillatti et al., 2003) which forms part of the suppression cassette Intervening Sequence 6683 – 6693 Sequence used in DNA cloning

B-Right Border 6694 – 7024 DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA (Zambryski et al., 1982)

Vector Backbone Intervening Sequence 7025 – 7173 Sequence used in DNA cloning

OR- ori V 7174 – 7570 Origin of replication from the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium (Stalker et al., 1981). Intervening Sequence 7571 – 7656 Sequence used in DNA cloning

MLHG ASSRPATARK SSGLSGTVRI PGDKSISHRS FMFGGLASGE TRITGLLEGE DVINTGKAMQ AMGARIRKEG

DTWIIDGVGN GGLLAPEAPL DFGNAATGCR LTMGLVGVYD FDSTFIGDAS LTKRPMGRVL NPLREMGVQV KSEDGDRLPV

TLRGPKTPTP ITYRVPMASA QVKSAVLLAG LNTPGITTVI EPIMTRDHTE KMLQGFGANL TVETDADGVR TIRLEGRGKL

TGQVIDVPGD PSSTAFPLVA ALLVPGSDVT ILNVLMNPTR TGLILTLQEM GADIEVINPR LAGGEDVADL RVRSSTLKGV

TVPEDRAPSM IDEYPILAVA AAFAEGATVM NGLEELRVKE SDRLSAVANG LKLNGVDCDE GETSLVVRGR PDGKGLGNAS

GAAVATHLDH RIAMSFLVMG LVSENPVTVD DATMIATSFP EFMDLMAGLG AKIELSDTKA A

Figure IV-3. Deduced Amino Acid Sequence of the Mature CP4 EPSPS Protein in PV-GMPQ/HT4404 The amino acid sequence of the CP4 EPSPS protein was deduced from the full-length coding nucleotide sequence present in vector PV-GMPV/HT4404.Monsanto Company 09-SY-201U Page 59 of 471 V. GENETIC ANALYSIS

A multi-faceted approach was taken to characterize the genetic modification made to produce MON 87705. The results confirm that MON 87705 contains a single insert that is stably integrated and is inherited according to Mendelian principles over multiple generations. These conclusions were based on several lines of evidence: 1) Southern blot analyses to assay the entire soybean genome for the presence of DNA derived from the transformation plasmid PV-GMPQ/HT4404, and to confirm that a single copy of the cp4

epsps expression cassette and the FAD2-1A and FATB1-A suppression cassette was inserted at a single site and that the in sert is stably inheri ted; 2) DNA sequencing analyses to determine the exact sequence of the inserted DNA and allow a comparison to the T-DNA sequence in the transformation vect or to confirm that only the expected sequences were integrated; and 3) a compar ison of the genomic DNA flanking the insert to the sequence of the insertion site in conventional soybean to identify any rearrangements that occurred at the insertion site during tr ansformation. Taken together, the characterization of the gene tic modification demonstrates that a single copy of the TDNA was inserted at a singl e locus of the genome.

Southern blot analyses were used to dete rmine the number of copies and the insertion sites of the integrated DNA as well as th e presence or absence of plasmid backbone sequence. The Southern blot strategy was de signed to ensure that all potential transgenic segments would have been identified. The entire soybean genome was assayed with probes that spanned the complete transformati on plasmid to detect the presence of the

insertion as well as confirm the absen ce of any backbone sequence. This was accomplished by using probes that were less than 2 kb in length ensuring a high level of sensitivity. This high level of sensitivity wa s demonstrated for each blot by detection of a positive control added at the equivalent of 1/10 th of a genome. Two restriction enzyme sets were specifically chosen to independently conf irm the presence of the insert. This two enzyme design also maximizes the possibilit y of detecting an insertion elsewhere in the genome which could be missed if that ba nd comigrated with an expected band. Additionally, the restriction enzyme sets were chosen such that at least one enzyme from

each set resides in the known 5 ′ or 3 ′ flanking sequence and that together the enzyme sets result in overlapping segments covering the enti re insert. Therefore, at least one segment for each flank is of a predictable size and overl aps with another predic table size segment. This overlapping strategy confirms that the entire insert sequence is identified in a predictable hybridi zation pattern.

The results of these analyses of MON 87705 show that a single copy of the T-DNA is inserted at a single locus of the genome. Generational stability analysis demonstrated that an expected Southern blot fingerpri nt of MON 87705 has been maintained through four generations of the breedi ng history, thereby confirming th e stability of the insert. Results from segregation analyses show heritability and stabil ity of the insert occurred as expected across multiple generations, which co rroborates the molecular insert stability analysis and establishes the genetic behavior of the DNA insert at a single chromosomal

locus.

For each digest used to confirm copy num ber there were duplicated samples that consisted of equal amounts of digested DNA. One set of samples was run for a longerMonsanto Company 09-SY-201U Page 60 of 471 period of time (long run) than the second set (short run). The long run allows for greater resolution of large molecular weight DNA, wher eas the short run allows the detection of small molecular weight DNA. For estimating the sizes of bands present in the long run lanes of Southern blots, the mo lecular weight markers on the le ft of the figure were used. For estimating the sizes of bands present in the short run lanes, the molecular weight markers on the right of the figure were used.

The DNA sequencing analyses complement the Southern analyses. Southern analyses

determined that MON 87705 contains T-DNA I and T-DNA II-derived sequences at a single insertion site. Sequencing of the in sert and the flanking genomic DNA confirmed the organization of the elements within the insert, determined the 5' and 3' insert-to-plant junctions, determined the complete DNA seque nce of the insert and adjacent soybean genomic DNA, and confirmed that the geno mic DNA sequences flanking the 5' and 3'

ends of the insert in MON 87705 are native to the soybean genome. Each genetic element is intact and the sequence of the inse rt matches the corr esponding sequence in PV-GMPQ/HT4404. In addition, genomic rea rrangements at the in sertion site were assessed by comparing the insert and fla nking sequence to the insertion site in conventional soybean.

The stability of the DNA in sert across multiple genera tions (R3-R6) was also demonstrated by Southern blot fingerprint analyses. F our generations of MON 87705 were digested with one of the enzyme sets used for the copy number analysis and were hybridized with probes that woul d detect restriction segments that encompass the entire insert (two hybridization bands ). This fingerprint stra tegy consists of two border segments that assess not only th e stability of the insert, but also the stability of genomic DNA directly adjacent to the insert.

The Southern blot analysis confirmed the in sert reported in Figur e V-1 represents the only detectable insert in MON 87705. The genetic elements integrated in MON 87705 are summarized in Table V-2. Maps of pl asmid vector PV-GMPQ/HT4404, used in the transformation to produce MON 87705 and annot ated with the prob es used in the Southern analysis are presented in Figures IV-1 and IV-2. Figure V-1 shows a linear map depicting restriction si tes within the insert as well as within the known soybean genomic DNA immediately flanking the in sert in MON 87705. Based on the linear map of the insert and the plasmid map, a table summarizing the expected DNA segments for Southern analyses is presented in Table V- 1. In some of the Southern blots, the migration of the genomic DNA is slightly di fferent when compared to the migration of the molecular weight markers. These altered migrations are likely the result of different base pair composition and/or differences in salt concentration between the genomic DNA samples and the molecular weight marker (Sambrook, 1989). The generations used in these studies are depicted in the breeding hi story shown in Figure V- 7. Materials and

methods used for characterization of the in sert for MON 87705 are found in Appendix B.Monsanto Company 09-SY-201UPage 61 of 471Figure V-1. Schematic Representation of the Insert and GenomicFlanking Sequences in MON 87705A linear map of the insert and genomic DNAflanking the insert in MON87705 is shown. Identified on the map are genetic elementswithin the insert, as well as restrictionsites with positions relative to the size ofthe linear map for enzymes used in the Southernanalyses. Shown on the lower portion of the map are the expectedsizes of the DNA segments after digestion with respectiverestriction enzymes. Arrowheads ( ) indicate the end of the insert and the beginningof soybean genomic flanking sequence. Thearrows (→) indicated the sequence direction of the elements in MON 87705. The * indicates partial sequences from the Left Borderand Right Border sequences after integration into MON 87705 (Table V-2). Base pairs 906-3279 inthe 5’ flanking genomic DNA,and 10535-12908 in the 3’ flanking genomic DNA represent duplicated bases from the 3′end of the flanking soybean genomic DNA(see Section V.C).Monsanto Company 09-SY-201UPage 62 of 471Table V-1. Summary Chart of the ExpectedDNA Segments Based on Hybridizing Probes and Restriction Enzymes Used inMON 87705 AnalysisSouthern blotFigureV-2 V-3 V-4 V-5 V-6 V-8Probes Used inAnalysis1, 4, 6 2, 5 3 7, 9 8, 10 1, 6Positive Hybridization ControlsXhoI +NcoIDigestedPlasmid~3.2, ~9.9 kb ~3.2, ~9.9 kb ~3.2 kb ~9.9 kb ~9.9 kb ~9.9 kbProbe Templates1~1.8, ~2.1, and ~1.1 kb ~0.8 and ~1.0 kb~~2~1.3 and ~1.5 kb ~1.9 and ~0.6 kb ~1.8 and ~1.1 kbMON 87705 DNA DigestionNcoI~4.0 and ~5.7 kb ~4.0 and ~5.7 kb ~5.7 kb No band No band ~4.0 and ~5.7 kbSpeI> 8.1*and ~5.0kb > 8.1*and ~5.0kb > 8.1*kb No band No band--31probe templates were added to predigested conventional soybeanDNA when multiple probes are used in Southern blot analysis.2‘~~’ indicates that the plasmid template was the only positivecontrol used, because the Southern blot was hybridized with oneprobe.3‘--’ indicates that the particular restriction enzyme was not used in the analysis.*Southern analysis indicates this segment to be ~ 11 kb.Monsanto Company 09-SY-201U Page 63 of 471 Table V-2. Summary of Genetic Elements in MON 87705

Genetic Element 1 Location in Sequence 2 Function (Reference)

Unique 5' flanking sequence of the insert 1 – 905 Soybean genomic DNA

Sequence flanking 5' end

of the insert 906-3279 2374 bp of soybean genomic DNA duplicated from the 3' end of the flanking sequence of the insert

B 3 -Left Border 3280 – 3538 259 bp sequence from the B-Left Border region remaining after integra tion (Barker et al., 1983)

Intervening Sequence 3539 – 3574 Sequence used in DNA cloning

P 4

- FMV/Tsf1 3575 – 4614 Chimeric promoter consisting of enhancer sequences from the promoter of the Figwort Mosaic virus 35S RNA (Richins et al., 1987) combined with the promoter from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 alpha (Axelos et al., 1989)

L 5

- Tsf1 4615 – 4660 5' untranslated leader (exon 1) from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 al pha (Axelos et al., 1989)

I 6

- Tsf1 4661 – 5282 Intron with flanking exon sequence from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 al pha (Axelos et al., 1989)

Intervening Sequence 5283 – 5291 Sequence used in DNA cloning

TS 7

- CTP2 5292 – 5519 Targeting sequence from the ShkG gene encoding the transit peptide region of Arabidopsis thaliana EPSPS (Klee et al., 1987) th at directs transport of the CP4 EPSPS protein to the chloroplast

CS 8

- cp4 epsps 5520 – 6887 Codon modified coding sequence of the aroA gene from the Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein (B arry et al., 1997; Padgette et al., 1996c)

Intervening Sequence 6888 – 6945 Sequence used in DNA cloning

3 B

– Border; P 4

– Promoter; L 5

– Leader; I 6

– Intron; TS 7

– Targeting Sequence; CS 8

– Coding Sequence;Monsanto Company 09-SY-201U Page 64 of 471 Table V-2 (cont.) Summary of Genetic Elements in MON 87705

Genetic Element Location in Sequence Function (Reference)

T 9

- E9 6946 – 7588 3' untranslated region of the pea RbcS2 gene which functions to direct pol yadenylation of the mRNA (Coruzzi et al., 1984)

Intervening Sequence 7589 – 7676 Sequence used in DNA cloning

P -7S α′ 7677 – 8517 Promoter and leader from the Sphas1 gene of Glycine max encoding beta-conglycinin storage protein (alpha'-bcsp) (D oyle et al., 1986) that directs transcription in seed

Intervening Sequence 8518 – 8539 Sequence used in DNA cloning

FAD2-1A p 8540 – 8805 Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 desaturase (Fillatti et al., 2003) which forms part of the suppression cassette

FATB1-A p 8806 - 9106 Partial sequence from the 5' untranslated region and the plastid targeting sequence from Glycine

max FATB1-A gene that encodes the palmitoyl acyl carrier protein thioesterase (Fillatti et al., 2003) which forms part of the suppression cassette

Intervening Sequence 9107 - 9114 Sequence used in DNA cloning

B-Right Border 9115 – 9134 20 bp sequence from the B-Right Border region remaining after integration (Zambryski et al., 1982)

B-Left Border 9135 - 9172 38 bp sequence from the B-Left Border region remaining after integra tion (Barker et al., 1983)

FATB1-A p1 9173 – 9443 Partial sequence from the 5' untranslated region and the plastid targeting sequence from Glycine

max FATB1-A gene that encodes the palmitoyl acyl carrier protein thioesterase (Fillatti et al., 2003) which forms part of the suppression cassette

FAD2-1A p 9444 – 9709 Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 desaturase (Fillatti et al., 2003) which forms part of the suppression cassette

Intervening Sequence 9710 – 9721 Sequence used in DNA cloning

T 9

– 3' untranslated transcriptional termination sequence and polyadenylation signal sequences; P – Partial

sequence; P1 – Truncated partial sequence of FATB1-A

.Monsanto Company 09-SY-201U Page 65 of 471 Table V-2 (cont.) Summary of Genetic Elements in MON 87705

Genetic Element Location in Sequence Function (Reference)

T-

H6 9722 – 10157 3' UTR sequence of the H6 gene from Gossypium

barbadense encoding a fiber pr otein involved in secondary cell wall assembly (John and Keller, 1995)

Intervening Sequence 10158 –10255 Sequence used in DNA cloning

B-Left Border 10256 - 10530 275 bp sequence from the B-Left Border region remaining after integration (Adang et al., 1985)

Sequence flanking 3' end

of the insert 10531 – 12908 Soybean genomic DNA including the 2374 bases duplicated at the 5' end of the flanking sequence

of the insert

Unique 3' flanking sequence of the insert 12909 – 13243 Soybean genomic DNA

V.A. Copy Number of T-DNA I and T-DNA II in MON 87705

The copy number and insertion sites of T-DNA I and T-DNA II were assessed by digesting test DNA with restriction enzymes Nco I or Spe I and hybridizing Southern blots with probes that span T-DNA I and T-DNA II (Figures IV-1 and IV-2). Each restriction digest is expected to produce a specifi c banding pattern on the Southern blots (Table V-1). Since each detected segm ent contains flanking genomic DNA, any additional integrated sites would produce a different bandi ng pattern with additional

bands.

The restriction enzyme Nco I cuts once in the MON 87705 in sert and once in each of the known 5' and 3' flanking sequences of MON 87705. Therefore, if T-DNA I and T-DNA II sequences are present at a single inte gration site in MON 87705, the digestion

with Nco I was expected to generate two border segments with expected sizes of ~4.0 kb and ~5.7 kb (Figure V-1). The ~4.0 kb restriction segment contains genomic DNA flanking the 5' end of the insert, the Left Border, the FMV/Tsf1 promoter, the Tsf1 leader,

and the Tsf1 intron. The ~5.7 kb restri ction segment contains the CTP2 targeting

sequence, cp4 epsps coding sequence, E9 3' untranslated sequence, 7S α '

promoter, FAD2-

1A and FATB1-A sense sequences, partial sequences of Right Border and Left Border,

FATB1-A and FAD2-1A antisense sequences, H6 3' untranslated sequence, Left Border and genomic DNA flanking the 3' end of the insert.

The restriction enzyme Spe I cuts once in the MON 87705 insert and once in the known 3' flanking sequence of MON 87705. Therefore, if T-DN A I and T-DNA II sequences are present at a single integrati on site in MON 87705 digestion with Spe I is expected to release two border segments with expected sizes of ~5.0 kb and greater than 8.1 kb (Figure V-1). Since the Spe I site in the soybean genome flan king the 5' end of the insert lies outside of the known sequen ce, it was not possible to pred ict a precise segment size.Monsanto Company 09-SY-201U Page 66 of 471 However, the segment size was determined by Southern blot anal yses to be ~11 kb (Figures V-2, V-3, and V-4). The ~11 kb DNA segment contains genomic DNA flanking the 5' end of the insert, Left Border, FMV/Tsf1 promoter, Tsf1 leader, Tsf1 intron, CTP2 targeting sequence, cp4 epsps coding sequence, E9 3' untranslated sequence, and a portion of 7S α '

promoter. The ~5.0 kb restriction segment contains the remaining portion

of the 7S α '

promoter, FAD2-1A and FATB1-A sense sequences, partial sequences of Right Border and Left Border, FATB1-A and FAD2-1A antisense sequences, H6 3' untranslated sequence, Left Border and genom ic DNA flanking the 3' end of the insert.

In the Southern blot analyses performed, each Southern blot contained a negative and several positive controls. Conven tional soybean DNA digested with Nco I or Spe I was used as a negative control to determine if the probes hybridized to any endogenous soybean sequences. As a positive control on the Southern blots, digested plasmid and probe templates were used. Plasmid PV-G MPQ/HT4404 digested with a combination of

Xho I and Nco I was mixed with predigested c onventional soybean genomic DNA and loaded on the gel. For Southern blots hybridized with multiple probes, each probe template was mixed with predigested conventional soybean DNA. The positive hybridization control was spiked at 0.1 and 1 genom e equivalent to demonstrate sufficient sensitivity of the Southern blot. Individual Southern blots were hybridized with the following probe sets: Probes 1, 4, a nd 6; Probes 2 and 5; Probe 3; Probes 7 and 9; and Probes 8 and 10 (refer to Figures IV-1 and 2 and Table V-1). The results of this analysis are shown in Fi gures V-2 through V-6.

V.A.1. Probes 1, 4 and 6

Conventional soybean DNA digested with Nco I (Figure V-2, lanes 1 and 8) or Spe I (Figure V-2, lanes 3 and 10) and hybridized w ith the probes 1, 4, and 6 (Figure IV-1) produced several hybridization signals. Th ese hybridization signals result from the probes (Probes 1, 4, and 6, Figure IV-1) hybr idizing to endogenous sequences residing in the soybean genome and are not specific to the inserted DNA. These results were expected, because several genetic elements c overed by probes 1, 4 and 6 are native to the soybean genome. These signals, as expe cted, were produced in both test and conventional soybean lanes, and therefore th e bands are considered to be endogenous background hybridization.

Probe template spikes (Probes 1, 4 and 6, Figure IV-1) generated from plasmid PV-GMPQ/HT4404 were mixed with the conv entional soybean DNA predigested with Spe I and produced the expected bands at ~1.8 , ~2.1, and ~1.1 kb, respectively, (Figure V-2, lanes 5-6) in addition to the endoge nous background hybridization observed in the conventional soybean DNA (Figure V-2, lane 10). Plasmid PV-GMPQ/HT4404 digested

with a combination of Xho I and Nco I and mixed with c onventional soybean DNA predigested with Spe I (Figure V-2, lane 7) produced the expected size bands of ~3.2 and ~9.9 kb (refer to Figure V-1) in additi on to the endogenous background hybridization observed in the conventional soybean DNA (Fi gure V-2, lane 10). These results indicate that the probes are hybridizi ng to their target sequences.

MON 87705 DNA digested with Nco I (Figure V-2, lanes 2 and 9) produced two unique

bands of ~4.0 and ~5.7 kb in addition to the endogenous background hybridizationMonsanto Company 09-SY-201U Page 67 of 471 observed in the conventional soybean DNA (F igure V-2, lanes 1 and 8). The ~4.0 kb band is the expected size for the border segm ent containing the 5' end of the inserted DNA (T-DNA I) along with the adjacent genomic DNA flanking the 5' end of the insert (Figure V-1). The ~5.7 kb band is the expect ed size for the border segment containing the 3' end of the inserted DNA (T-DNA I and II) along with the adjacent genomic DNA flanking the 3' end of th e insert (Figure V-1).

MON 87705 DNA digested with Spe I (Figure V-2, lanes 4 and 11) produced two unique bands of ~5.0 and ~11 kb, in addition to the endogenous background hybridization observed in the conventional soybean DNA (Fig ure V-2, lanes 3 and 10). The ~11 kb segment is consistent with the expected band being greater than 8.1 kb. This band in the short run appears slightly larg er, at ~13 kb, than the corres ponding band in the long run. This border segment contains the 5' end of the inserted DNA (T-DNA I) along with the adjacent genomic DNA flanking the 5' end of th e insert (Figure V-1). The ~5.0 kb band (Figure V-2, lane 4) is consistent with the expected band of 5.0 kb; however, the migration of the segment is slightly higher at ~5.2 kb in the short run (Figure V-2, lane 11) as indicated by the molecular weight marker. The ~5.0 kb band is the expected size for the border segment containing the 3' end of the inserted DNA (T-DNA I and II) along with the adjacent genomic DNA flanking the 3' end of the insert (Figure V-1).

There were no additional bands detected usi ng the probes 1, 4, and 6. Based on the results presented in Figure V-2, it was concluded that T-DNA sequences covered by probes 1, 4, and 6 reside at a single integrati on locus in MON 87705.

V.A.2. Probes 2 and 5

Conventional soybean DNA digested with Nco I (Figure V-3, lanes 1 and 8) or Spe I (Figure V-3, lanes 3 and 10) and hybridized with probes 2 and 5 (Figure IV-1) produced several hybridization signals. These hybridiz ation signals result from the probes (Probes 2 and 5, Figure IV-1) hybridizing to endoge nous sequences residing in the soybean genome and are not specific to the inserted DNA. These results were expected, because several genetic elements covered by probes 2 and 5 are native to the soybean genome. These signals, as expected, were produced in both test and conventional soybean lanes,

and therefore the bands are consider ed to be endogenous background.

Probe template spikes (Probes 2 and 5, Figure IV-1) generated from plasmid PV-GMPQ/HT4404 mixed with the conventio nal soybean DNA predigested with Spe I produced the expected bands at ~0.8 and ~1.0 kb (Figure V-3, lanes 5-6) in addition to the endogenous background hybridization obse rved in the conventional soybean DNA (Figure V-3, lane 10). Plasmid PV-GMP Q/HT4404 digested with a combination of Xho I

and Nco I and mixed with conventiona l soybean DNA predigested with Spe I (Figure V3, lane 7) produced the expected size bands of ~3.2 and ~9.9 kb (refer to Figure V-1) in addition to the endogenous background hybridization observ ed in the conventional soybean DNA (Figure V-3, lane 10). These results indicate that the probes are hybridizing to their target sequences.

MON 87705 DNA digested with Nco I (Figure V-3, lanes 2 and 9) produced two unique bands of ~4.0 and ~5.7 kb (Figure V-3, lane s 1 and 8). The ~4.0 kb band is the expected size for the border segment containing the 5' end of the inserted DNA (T-DNA I) alongMonsanto Company 09-SY-201U Page 68 of 471 with the adjacent genomic DNA flanking the 5' end of the insert (Figure V-1). The ~5.7 kb band is the expected size fo r the border segment containing the 3' end of the inserted DNA (T-DNA I and T-DNA II) along with the ad jacent genomic DNA flanking the 3' end of the insert (Figure V-1).

MON 87705 DNA digested with Spe I (Figure V-3, lanes 4 and 11) produced two unique bands of ~5.0 and ~11 kb (Figure V-3, lanes 3 and 10). The ~11 kb segment is consistent with the expected band being greater than 8.1 kb and with the ~11 kb segment seen with probes 1, 4, and 6 (Figure V-2, lanes 4 and 11). The ~11 kb band is the expected size for the border segment containing the 5' end of the inserted DNA (T-D NA I) along with the adjacent genomic DNA flanking the 5' end of the insert (Figur e V-1). The ~5.0 kb border segment contains the 3' end of the inse rted DNA (T-DNA I and T-DNA II) along with the adjacent genomic DNA flanking the 3' end of the insert (Figure V-1). There were no additional bands detected using probes 2 and 5. Based on th e results presented in Figure V-3, it was concluded that sequence cove red by probes 2 and 5 resides at a single integration locus in MON 87705.

V.A.3. Probe 3

Conventional soybean DNA digested with Nco I (Figure V-4, lanes 1 and 7) or Spe I (Figure V-4, lanes 3 and 9) and hybridized with probe 3 (Figure IV-1) showed no detectable hybridization bands, as expected for th e negative control.

Plasmid PV-GMPQ/HT4404 digested with a combination of Xho I and Nco I and mixed with conventional soybean DNA predigested with Spe I (Figure V-4, lanes 5-6) produced the expected size band of ~3.2 kb (refer to Fi gure V-1). This hybridi zation indicates that the probe is hybridizing to its target sequence.

MON 87705 DNA digested with Nco I (Figure V-4, lanes 2 and 8) produced the expected band of ~5.7 kb. The ~5.7 kb band is the expe cted size for the bor der segment containing the 3' end of the inserted DNA (T-DNA I and T-DNA II) along with the adjacent genomic DNA flanking the 3' end of the insert (Figure V-1).

MON 87705 DNA digested with Spe I (Figure V-4, lanes 4 and 10) produced the expected band of ~11 kb. The ~11 kb band is consistent with the expected band being greater than 8.1 kb and with the ~11 kb segment seen in Fi gures V-2 and 3 (lanes 4 and 11). The ~11 kb band represents the border segment containing the 5' end of the inserted DNA (T-DNA I) along with the adjacent genomic DNA flanking the 5' end of the insert (Figure V-1).

There were no additional bands detected using probe 3. Based on the results presented in Figure V-4, it was concluded that sequen ce covered by probe 3 resides at a single detectable integration locus in MON 87705.

Taken together, the data pres ented in Figures V-2, V-3, and V-4 indicate that a single copy of the T-DNA I and T-DNA II sequences in tegrated into the soybean genome at a single detectable site in MON 87705.Monsanto Company 09-SY-201U Page 69 of 471 Figure V-2. Southern Blot Analysis of MON 87705: Probes 1, 4, and 6
The blot was hybridized with 32 P-labeled probes that span a portion of T-DNA I and T-DNA II sequences (probes 1, 4, and 6, Figure IV-1). Each lane contains ~10 μ g of digested genomic DNA isolated from leaf tissue. Lane designations are as follows: Lane 1: Conventional soybean ( Nco I) 2: MON 87705 ( Nco I) 3: Conventional soybean ( Spe I) 4: MON 87705 ( Spe I) 5: Conventional soybean ( Spe I) spiked with probe templates [~0.1 genomic equivalent] 6: Conventional soybean ( Spe I) spiked with probe templates [~1 genomic equivalent] 7: Conventional soybean ( Spe I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~1 genomic equivalent] 8: Conventional soybean ( Nco I) 9: MON 87705 ( Nco I) 10: Conventional soybean ( Spe I) 11: MON 87705 ( Spe I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.Monsanto Company 09-SY-201U Page 70 of 471 Figure V-3. Southern Blot Analysis of MON 87705: Probes 2 and 5
The blot was hybridized with 32 P-labeled probes that span a portion of T-DNA I sequences (probes 2 and 5, Figure IV-1). Each lane contains ~10 μ g of digested genomic DNA isolated from leaf tissue. Lane designations are as follows: Lane 1: Conventional soybean ( Nco I) 2: MON 87705 ( Nco I) 3: Conventional soybean ( Spe I) 4: MON 87705 ( Spe I) 5: Conventional soybean ( Spe I) spiked with probe templates [~0.1 genomic equivalent] 6: Conventional soybean ( Spe I) spiked with probe templates [~1 genomic equivalent] 7: Conventional soybean ( Spe I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~1 genomic equivalent] 8: Conventional soybean ( Nco I) 9: MON 87705 ( Nco I) 10: Conventional soybean ( Spe I) 11: MON 87705 ( Spe I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium st ained gel.Monsanto Company 09-SY-201U Page 71 of 471 Figure V-4. Southern Blot Analys is of of MON 87705: Probe 3
The blot was hybridized with a 32 P-labeled probe that span a portion T-DNA I sequences (probe 3, Figure IV-1). Each lane contains ~10 μ g of digested genomic DNA isolated from leaf tissue. Lane designations

are as follows: Lane 1: Conventional soybean ( Nco I) 2: MON 87705 ( Nco I) 3: Conventional soybean ( Spe I) 4: MON 87705 ( Spe I) 5: Conventional soybean ( Spe I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~0.1 genomic equivalent] 6: Conventional soybean ( Spe I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~1 genomic equivalent] 7: Conventional soybean ( Nco I) 8: MON 87705 ( Nco I) 9: Conventional soybean ( Spe I) 10: MON 87705 ( Spe I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.Monsanto Company 09-SY-201U Page 72 of 471 V.B. Southern Blot Analysis to Determ ine the Presence or Absence of Plasmid PV-GMPQ/HT4404 Backbone

MON 87705 and conventional soybean genomic DNA were digested wi th the restriction

enzymes Nco I or Spe I. Probe template spikes (pr obes 7-10, Figure IV-2) generated from plasmid PV-GMPQ/HT4404 were mixed w ith the predigested conventional soybean genomic DNA to serve as positive hybridi zation controls. Additionally, plasmid PV-GMPQ/HT4404 DNA previously dige sted with the combination of Xho I and Nco I

was mixed with conventional soybean DNA digested with Spe I and loaded on the gel to serve as a positive hybridization control. Th e blots were hybridized with probes 7-10 (Figure IV-2) that covered the entire b ackbone sequence of PV-GMPQ/HT4404. If backbone sequences are present in MON 87705, then probing with backbone sequence should result in unique hybridizing bands. Th e results are shown in Figures V-5 and V-6.

V.B.1. Plasmid Backbone Probes 7 and 9

Conventional soybean DNA digested with the restriction enzyme Nco I (Figure V-5, lanes 1 and 8) or Spe I (Figure V-5, lanes 3 and 10) and hybridized with probes 7 and 9 (Figure IV-2) showed no dete ctable hybridization bands, as expected for the negative

control.

Probe template spikes (Probes 7 and 9, Figure IV-2) generated from plasmid PV-GMPQ/HT4404 and mixed with conven tional soybean DNA predigested with Spe I produced the expected bands at ~1.3 and ~1 .5 kb (Figure V-5, lanes 5 and 6). In addition, there is an unexpected faint band at ~3.0 kb (Figure V-5, lane 6). Based on size, this band is likely derived from dimers of the probe template (Qiagen at www.qiagen.com ). Since this extra band only is pres ent in the positive control and is not present in the conventional or MON 87705 DNA, it was concluded that the presence of this extra band did not impact or alter the final results for MON 87705. Plasmid PV-GMPQ/HT4404 digested with Xho I /Nco I and mixed with conventional soybean DNA digested with Spe I (Figure V-5, lane 7) produced an expected band that migrated at

~9.4 kb. This band is consistent with th e expected band at ~9.9 kb; however, the migration of the ~9.4 kb segments is slight ly lower than indicated by molecular weight marker most likely due to differences in salt concentrations between sample and marker. Overall, these results indicate that the probes are hybridizing to thei r target sequences.

MON 87705 DNA digested with either Nco I (Figure V-5, lanes 2 and 9) or Spe I (Figure V-5, lanes 4 and 11) showed no det ectable hybridization si gnal, indicating that MON 87705 does not contain any detectable backbone sequence from the transformation vector PV-GMPQ/HT4404 that is covered by probes 7 and 9.

V.B.2. Plasmid Backbone Probes 8 and 10

Conventional soybean DNA digested with the restriction enzyme Nco I (Figure V-6, lanes 1 and 8) or Spe I (Figure V-6, lanes 3 and 10) and hybridized with probes 8 and 10Monsanto Company 09-SY-201U Page 73 of 471 (Figure IV-2) showed no dete ctable hybridization bands, as expected for the negative

control.

Probe template spikes (Probes 8 and 10, Figure IV-2) generated from plasmid PV-GMPQ/HT4404 and mixed with conven tional soybean DNA predigested with Spe I produced two expected bands that migrated at ~0.7 and ~2.1 kb (Figure V-6, lanes 5 and 6). Plasmid PV-GMP Q/HT4404 digested with Xho I /Nco I and mixed with conventional soybean DNA digested with Spe I (Figure V-6, lane 7) produced an expected band that migrated at ~9.4 kb, whic h is consistent with the expected band at ~9.9 kb. The migration of the positive hybridi zation controls (Figure V-6, lanes 5-7) is slightly different than indi cated by the molecular weight marker, most likely due to

differences in salt concentrations between sa mples and markers. Overall, these results indicate that these prob es are hybridizing to thei r control sequences.

MON 87705 DNA digested with either Nco I (Figure V-6, lanes 2 and 9) or Spe I (Figure V-6, lanes 4 and 11) showed no detect able hybridization signal . These results in combination with Figure V-5 indicate that MON 87705 does not cont ain any detectable backbone sequence from the tran sformation vector PV-GMPQ/HT4404.Monsanto Company 09-SY-201U Page 74 of 471 Figure V-5. Southern Blot Analysis of MON 87705:PV-GMPQ/HT4404 Backbone

Probes 7 and 9
The blot was hybridized with 32 P-labeled probes that span a portion of backbone sequences (probes 7 and 9, Figure IV-2) of plasmid PV-GMPQ/HT4404. Each lane contains ~10 μ g of digested genomic DNA

isolated from leaf tissue. La ne designations are as follows: Lane 1: Conventional soybean ( Nco I) 2: MON 87705 ( Nco I) 3: Conventional soybean ( Spe I) 4: MON 87705 ( Spe I) 5: Conventional soybean ( Spe I) spiked with probe templates [~0.1 genomic equivalent] 6: Conventional soybean ( Spe I) spiked with probe templates [~1 genomic equivalent] 7: Conventional soybean ( Spe I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~1 genomic equivalent] 8: Conventional soybean ( Nco I) 9: MON 87705 ( Nco I) 10: Conventional soybean ( Spe I) 11: MON 87705 ( Spe I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.Monsanto Company 09-SY-201U Page 75 of 471 Figure V-6. Southern Blot Analys is of MON 87705: PV-GMPQ/HT4404

Backbone Probes 8 and 10
The blot was hybridized with 32 P-labeled probes that span a portion of backbone sequences (probes 8 and 10, Figure IV-2) of plasmid PV-GMPQ/HT4404. Each lane contains ~10 μ g of digested genomic DNA

isolated from leaf tissue. La ne designations are as follows: Lane 1: Conventional soybean ( Nco I) 2: MON 87705 ( Nco I) 3: Conventional soybean ( Spe I) 4: MON 87705 ( Spe I) 5: Conventional soybean ( Spe I) spiked with probe templates [~0.1 genomic equivalent] 6: Conventional soybean ( Spe I) spiked with probe templates [~1 genomic equivalent] 7: Conventional soybean ( Spe I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~1 genomic equivalent] 8: Conventional soybean ( Nco I) 9: MON 87705 ( Nco I) 10: Conventional soybean ( Spe I) 11: MON 87705 ( Spe I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.Monsanto Company 09-SY-201U Page 76 of 471 V.C. Organization and Sequence of the Insert and Adjacent Genomic DNA in

MON 87705

The organization of the elements within the MON 87705 insert was confirmed by DNA sequence analyses. Several PCR primers were designed with the intent to amplify six overlapping regions of DNA that span the en tire length of the insert (see Appendix B). The amplified DNA segments were subjected to DNA sequencing analyses. The insert in MON 87705 is 7251 base pairs and matche s the sequence of PV-GMPQ/HT4404 as

described in Tables IV-3 and V-2.

A sequence comparison between the PCR pr oduct generated from the conventional soybean (A3525) and the sequence generated fr om the 5' and 3' flanking sequences of MON 87705 indicates there was a 36 bp deleti on (bases 896-931) and a 2374 bp insertion just 5' to the MON 87705 in sertion site. Given the ve ry high homology between the 2374 bases flanking the 5’ end of the insert and the genomic DNA flanking the 3’ end of the insert, the 2374 bases are most likely from the 3' end of the flanking genomic DNA and were duplicated at the 5' end of the insertion site when T-DNA I and T-DNA II integrated into the genome. This duplicati on has the following characteristics: 1) there is a single nucleotide change detected in the dupl icated 2374 base pairs at the 5' flanking sequence; and 2) there are four unique bases located at the junction of the insert DNA and the 2374 bases in the 3' flanking sequence. This analysis confirms that the genomic sequences flanking the insert in MON 87705 are native to the soybean genome and that a 36 base-pair deletion and a 2374 base pair duplica tion that contains a single base change occurred at the insertion site during in tegration of the T-DNA sequences. These molecular rearrangements presumably resu lted from double-stra nded break repair mechanisms in the plant during the Agrobacterium -mediated transformation process (Salomon and Puchta, 1998).

V.D. Southern Blot Analysis to Examine Insert Stability in Multiple Generations

of MON 87705

In order to demonstrate the stability of the T-DNA I and T-DNA II insert in MON 87705 in multiple generations, Southern blot an alyses were perfor med using DNA obtained from multiple generations of the MON 87705 breeding history. For reference, the breeding history of MON 87705 is presented in Figure V-7. The specific generations tested are indicated in the legends of Figure V-8. DNA samples from R3, R4, R5, and R6 generations of MON 87705 (refer to Figure V-7) were digested with Nco I and were expected to release two border segments with the expected sizes of 4.0 and 5.7 kb (Figure V-1). The detected hybridizat ion bands in R4, R5, and R6 generations are compared to the fully characterized MON 87705 R3 generati on to evaluate stabi lity. Any instability

associated with the insert would be detected as faint novel bands with in the fingerprint on

the Southern blot. The blot was hybridized simultaneously with two radiolabeled probes that cover both border segments generated by the digest (probes 1 and 6, Figure IV-1). This blot has two of the same positive hybridi zation controls (probes 1 and 6, Figure IV1) as described in Section V.A.1. The result of this analysis is shown in Figure V-8.Monsanto Company 09-SY-201U Page 77 of 471 Conventional soybean DNA digested with Nco I and hybridized with probes 1 and 6 (Figure V-8, lane 4) showed hybridization bands. These hybri dization signals result from the probes hybridizing to endoge nous targets residi ng in the soybean genome and are not specific to the inserted DNA.

Probe templates spikes (Probes 1 and 6, Figure IV-1) generated from plasmid PV-GMPQ/HT4404 and mixed with convent ional soybean DNA predigested with Nco I (Figure V-8, lanes 1 and 2) produced the exp ected size bands at ~1.8 and ~1.1 kb. The detection of the probe template positive hybrid ization controls demonstrates that both probes were hybridizing to the target DNA. Plasmid PV-GMPQ/HT4404 digested with a

combination of Xho I and Nco I and mixed with conventional soybean DNA predigested

with Nco I (Figure V-8, lane 3) produced the expected size band at ~9.9 kb, which indicates that the probes are hybridizi ng to their corresponding sequence in the transformation vector. This expected band at ~9.9 kb migrated together with an endogenous hybridization signal observe d in Figure V-8, lane 4.

Digestion of MON 87705 from multiple generations (refer to Breeding History of MON 87705, Figure V-7) with restriction enzyme Nco I produced two bands at ~4.0 and ~5.7 kb (Figure V-8, lanes 5-8) in add ition to the endogenous b ackground hybridization observed in the conventional soybean DNA (Figur e V-8, lane 4). The ~4.0 kb band is the expected size for the border segment contai ning the 5' end of the inserted DNA (T-DNA I) along with the adjacent genom ic DNA flanking the 5' end of the insert (Figure V-1). The ~5.7 kb band is the expected size for th e border segment containing the 3' end of the inserted DNA (T-DNA I and T- DNA II) along with the adjacen t genomic DNA flanking the 3' end of the insert (Figure V-1). Howe ver, the migration of this segment appears slightly lower than indicated by the molecu lar weight marker most likely due to

differences in salt concentrations between the samples and marker. This restriction pattern is the same as the restriction pattern observed in the Southern blot analysis of the R3 generation shown in Figur e V-2 (lanes 2 and 9).

There were no additional unexpect ed bands detected, indicati ng that the single copy of T-DNA I and T-DNA II in MON 87705 is stably ma intained in the selected generations.Monsanto Company 09-SY-201U Page 78 of 471 Figure V-7. Breeding History of MON 87705 All generations were self pollinated ( ⊗ ) . The R3 generation was used for the molecular analyses of MON 87705 reporte d in Figures V-2 through V-6 and is referred to as MON 87705 in all Southern blot figures. The R3, R4, R5, and R6 generations were used for analyzing the stability of the insert in multiple generations. 

# ⊗Monsanto Company 09-SY-201U Page 79 of 471 Figure V-8. Generational Stabilit y of MON 87705: Probes 1 and 6
The blot was hybridized with 32 P-labeled probes that spanned a portion of T-DNA I and T-DNA II sequences (probes 1, and 6, Figure IV-1). Each lane contains ~ 10 μ g of digested genomic DNA isolated from leaf tissue. The breeding history of MON 87705 is illustrated in Figure V-7. Lane designations are as

follows: Lane 1: Conventional soybean ( Nco I) spiked with probe templates [~0.1 genomic equivalent] 2: Conventional soybean ( Nco I) spiked with probe templates [~1 genomic equivalent] 3: Conventional soybean ( Nco I) spiked with PV-GMPQ/HT4404 ( Xho I /Nco I) [~1 genomic equivalent] 4: Conventional soybean ( Nco I) 5: MON 87705 [R3, ( Nco I)] 6: MON 87705 [R4, ( Nco I)] 7: MON 87705 [R5, ( Nco I)] 8: MON 87705 [R6, ( Nco I)] Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.Monsanto Company 09-SY-201U Page 80 of 471 V.E. Inheritance of the Genetic Insert in MON 87705

During development of MON 87705 segregati on data were recorded to assess the heritability and stability of the coding seque nces present in MON 87705. Chi-square analysis was performed over se veral generations to confirm the segregation and stability of the MON 87705 insertion. The Chi-square analysis is based on testing the observed segregation ratio to the exp ected segregation ratio accordi ng to Mendelian principles.

The MON 87705 breeding path from which segreg ation data were generated is described in Figure V-9. The transformed R0 plant wa s self-pollinated to pr oduce R1 seed. From the R1 segregating population, an i ndividual plant (#90, designated MON 87705) homozygous for a single copy of the H6 3’UTR was identified via Invader (Wave Technologies, Inc.) and Sout hern blot analysis.

The selected R1 MON 87705 plant was self-pollin ated to give rise to a population of R2 plants that were repeatedly self-pollinated through to the R4 generation. At each generation, the fixed homozygous plants were tested for the expected segregation pattern of 1:0 (positive: negative) for the H6 3’UTR using Invade r analysis.

At the R4 generation, homozygous MON 87705 plants were bred via traditional breeding with a soybean variety that did not contain the H6 3’UTR to produce F1 hemizygous seed. The resulting F1 plants were then self-pollinated to produce F2 seed. The heritability and stability of the coding se quences present in MON 87705 were assessed from plants of the F2, F3, F4, and F5 gene rations. At each of these generations, the plants were tested for the presence of the H6 3’UTR by Invader analysis, and hemizygous positive plants were then selected and self -pollinated to produce seed of the next generation.

A Chi-square ( χ 2 ) analysis was used to compare the observed segregation ratios to the expected ratios according to Mendelian princi ples. The Chi-square value was calculated

as:

χ 2 = ∑ [( | o – e | ) 2 / e]

where o = observed frequency of the genotype and e = e xpected frequency of the genotype. The level of statistical sign ificance was predetermined to be 5% (p ≤ 0.05).

The results of the χ 2 analysis of the segregating proge ny of MON 87705 are presented in

Table V-3. The χ 2 values in the F2 generation indi cated no statistically significant difference between the observed and expect ed 1:2:1 (homozygous positive:hemizygous positive:homozygous negative) segregation ratio. The χ 2 value in the F3 generation indicated a statistically signi ficant difference between the ob served and expected 1:2:1 segregation ratio. However, there were ten plan ts out of 91 total plants tested in the assay in which the zygosity could not be determined by Invader analysis. These missing data combined with a relatively small sample size (n=81) of tested plants may have skewed the segregation ratio. This caused the results of the analysis to be inconclusive and the data from the F3 generation c ould not be used to accurately assess the heritability and stability of the coding sequences present in MON 87705. Therefore, the F4 and F5Monsanto Company 09-SY-201U Page 81 of 471 generations were tested using larger sample sizes to further assess the heritability and stability of the inserted coding sequences. The χ 2 values in the F4 and F5 generations indicated no statistically si gnificant differences between th e observed and expected 1:2:1 segregation ratios. Considering the data fr om three generations (F2, F4, and F5), the results support the conclusion that the codi ng sequences present in MON 87705 reside at a single locus within the soybean genome and are inheri ted according to Mendelian inheritance principles. These results ar e also consistent with the molecular characterization data that indicate a singl e genomic insertion site for the coding sequences present in MON 87705 that encode for the improved fatty acid profile and glyphosate tolerance trait (Table VI-1).Monsanto Company 09-SY-201UPage 82 of 471Transformed and regenerated R0 plantR1R2(homozygous positive)R3 (homozygous positive)R4 (homozygous positive) F1 (hemizygous positive)Breeding path continued F2 (expected segregation of 1:2:1)1homozygous positive :hemizygous positive: homozygous negativeF3 (expected segregation of 1:2:1)1homozygous positive :hemizygous positive: homozygous negativeF4 (expected segregation of 1:2:1)1homozygous positive :hemizygous positive: homozygous negativeF5 (expected segregation of 1:2:1)1homozygous positive :hemizygous positive: homozygous negative⊗ =Self pollinatedFigure V-9. Breeding Path for Generating Segregation Data MON 877051Chi-square analysis conductedon segregation data from the F2, F3, F4 and F5 generations⊗Plant #90 (MON 87705) selected and self pollinated⊗⊗⊗cross with variety that didnot contain theH63’UTR⊗⊗⊗Monsanto Company 09-SY-201UPage 83 of 471Table V-3. Segregation of the H6 3’UTRGene During the Development of MON 877051:2:1 SegregationGeneration1TotalPlantsTested2Observed #PlantsHomozygousPositiveObserved #PlantsHemizygousPositiveObserved #PlantsHomozygousNegativeExpected #PlantsHomozygousPositiveExpected #PlantsHemizygousPositiveExpected #PlantsHomozygousNegativeχ2ProbabilityF241971009209110971049.252098.51049.253.70.1538F38130351620.2540.520.256.30.0421F426668 126 7266.513366.50.90.6514F517544884343.7587.543.750.00.99151F2 progeny were from the cross of MON 87705 homozygous positive for theH63’UTR with a soybean variety that did not contain theH63’UTR.F3, F4, and F5 progeny were from self-pollinated plants of the previous generation that werehemizygous positive for theH63’UTR.2Plants were tested for the presence of theH63’UTR by Invader analysis. “Total plants” refersto the total number of plants in which zygositycould be determined using the assay.Monsanto Company 09-SY-201U Page 84 of 471 V.F. Conclusion of Molecular Characterization

Molecular characterization of MON 87705 by Southe rn blot analyses demonstrated that a single copy of the T-DNA I and T- DNA II sequences from the transformation vector PV-GMPQ/HT4404 was integrated into the soyb ean genome at a single locus. There were no additional genetic elements, including backbone sequences, from the transformation vector PV-GMPQ/HT4404 detected , linked or unlinked to the intact DNA insert, in MON 87705.

PCR and DNA sequence analyses were pe rformed on MON 87705, which confirmed the organization of the elements within the insert , determined the 5' and 3' insert-to-plant junctions, determined the complete DNA seque nce of the insert and adjacent soybean genomic DNA sequence in MON 87705, and conf irmed that the genomic DNA sequences flanking the 5' and 3' ends of the insert in MON 87705 ar e native to the soybean genome. The PCR and DNA sequence analysis identified 36 bp of conventional soybean DNA sequence deleted at the insertion site in MO N 87705. Additionally, a 2374 bp duplication was identified in the 5' genomic flanking sequence of MON 87705. This duplicated sequence is likely from the 3 ′ flanking sequence of MON 87705 and it contains a single nucleotide change.

Generational stability analysis demonstrated that an expected Southern blot fingerprint of MON 87705 has been maintained through four generations of the breeding history, thereby confirming the stability of the insert . Results from segregation analyses show heritability and stability of the insert occurred as expected across multiple generations,

which corroborates the molecu lar insert stability analysis and establishes the genetic behavior of the DNA insert at a single chromosomal locus.Monsanto Company 09-SY-201U Page 85 of 471 VI. CHARACTERIZATION OF THE INTRODUCED CP4 EPSPS PROTEIN

IN MON 87705

As described in Section V, the MON 87705 insert contains a cp4 epsps expression

cassette and a FATB/FAD2 supression cassette. As the FATB/FAD2 cassette does not encode for any proteins, this section focuses on the characterization and safety of the CP4 EPSPS protein produced by MON 87705.

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is mediated by dsRNA molecules. Doubl e stranded RNAs are commonly found in eukaryotes, including plants , for endogenous gene suppression and are composed of nucleic acids. Nucleic acids have a long hist ory of safe consumption and are considered GRAS by the U.S. Food and Drug Administra tion. There is no evidence to suggest dietary consumption of RNA is associated wi th toxicity or allergenicity. Moreover, analysis of the DNA segments encoding this ds RNA showed that they lack the sequences required for translation initiati on and protein synthesis. Th e production of a protein from the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several biotechnology-derived plant pr oducts previously deregulated by APHIS were developed using RNA-based suppression mechanisms, incl uding virus-resistant papaya and squash, high oleic soybean, FLAVR SAVR tomatoes, and plum trees resistant to Plum pox virus. Based on this information, it is concluded that the inserted DNA and resulting dsRNA are safe and unlikely to produce a protein or po lypeptide. As a result, the RNA-based suppression technology used in MON 87705 poses no novel risks from a food, feed or environmental perspective (FDA, 1994; USDA-APHIS, 1994; USDA-APHIS, 1997; USDA-APHIS, 2006; USDA-APHIS, 2007).

The remainder of this section summarizes the assessment of the CP4 EPSPS protein produced in MON 87705 including: 1) the identity of the CP4 EPSPS protein from MON 87705; 2) demonstration of the e quivalence of the plant-produced and E. coliproduced CP4 EPSPS proteins used in laboratory and regulato ry safety evaluations; 3) the CP4 EPSPS protein expressi on levels in MON 87705 s oybean tissues; and 4) an allergenicity assessment for the CP4 EPSP S protein. Results indicate that the MON 87705-produced CP4 EPSPS pr otein is equivalent to E. coli- produced protein. Data also support a conclusion of safe cons umption based on several lines of evidence, all of which will be submitted to FDA as part of the pre-market consultation.

VI.A. Identity and Function of the CP4 EPSPS Protein from MON 87705

The 5-enolpyruvylshikimate-3-phosphate synt hase (EPSPS: EC2.5.1.19) family of enzymes is ubiquitous to plants and microorgani sms. EPSPS proteins have been isolated from both sources, and their properties have been extensively studied (Harrison et al., 1996; Haslam, 1993; Klee et al., 1987; Sc honbrunn et al., 2001; Steinrucken and Amrhein, 1984). The shikimate pathway and the EPSPS protein are absent in mammals, fish, birds, reptiles, and insects (Alibhai and Stallings, 2001). Th e bacterial and plant enzymes are mono-functional w ith a molecular weight of 44-48 kDa (Kishore et al., 1988). EPSPS proteins catalyze the transf er of the enolpyruvyl group from phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P), thereby yielding inorganic phosphate and 5-enolpyruvylshikimate -3-phosphate (EPSP) (AlibhaiMonsanto Company 09-SY-201U Page 86 of 471 and Stallings, 2001). Due to th e specificity of EPSPS for its substrates, the only known catalytic product generated is EPSP, which is the penultimate product of the shikimic acid pathway. Shikimic acid is a substrate for the biosynthesis of the aromatic amino acids (phenylalanine, tryptophan and tyrosine) and other aromatic molecules. It has been estimated that aromatic molecules, all of wh ich are derived from shikimic acid, represent 35% or more of the dry weight of a plant (Franz et al., 1997).

The EPSPS transgene in M ON 87705 is derived from Agrobacterium sp. strain CP4 ( cp4

epsps ). The cp4 epsps coding sequence encodes a 47.6 kDa EPSPS protein consisting of a single polypeptide of 455 amino acids (Padge tte et al., 1996b). The CP4 EPSPS protein is similar and functionally id entical to endogenous plant EP SPS enzymes, but has a much reduced affinity for glyphosate, the activ e ingredient in the Roundup family of agricultural herbic ides, relative to endogenous plant EPSPS (Padgette et al., 1996b). In conventional plants, glyphosate binds to th e endogenous plant EPSPS enzyme and blocks the biosynthesis of shikimate-3-phosphate, th ereby depriving plants of essential amino acids (Haslam, 1993; Steinrücken and Amrh ein, 1980). In Roundup Ready plants, which are tolerant to the Roundup family of agricu ltural herbicides, requirements for aromatic

amino acids and other metabolites are met by the continued action of the CP4 EPSPS enzyme in the presence of glyphosate (Padgette et al., 1996b ). The CP4 EPSPS protein expressed in MON 87705 is identical to the CP4 EPSPS protein in other Roundup Ready crops including Roundup Ready soybean (404 -3-2), Roundup Ready 2 Yield soybean (MON 89788), Roundup Ready Corn 2, Roundu p Ready canola, Roundup Ready sugar beet, and Roundup Ready cotton.

VI.B. Characterization of the Full Leng th CP4 EPSPS Protein from MON 87705

The safety assessment of crops derived th rough biotechnology incl udes characterization of the introduced protein produced from the inserted DNA, confirmation of its functional and physicochemical properties, and confirmation of the safety of the protein. The level of CP4 EPSPS protein produced in MON 87705 is too low to allow purification of sufficient quantities for use in subsequent sa fety assessment studies. Therefore, it is necessary to produce th e protein in high-expressing reco mbinant host systems (such as bacteria) in order to obtain sufficient qua ntities of the CP4 E PSPS protein. CP4 EPSPS protein was produced in E. coli , and subsequently purified and characterized. A small quantity of the CP4 EPSPS prot ein was also puri fied from harvested MON 87705 seed. The equivalence of the physicochemical char acteristics and functional activity between the MON 87705-produced and E. coli- produced CP4 EPSPS protei ns was confirmed by a panel of analytical technique s, including: (1) sodium dode cyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to establish equiva lence of the apparent molecular weight between MON 87705-produced and the E. coli -produced reference standard protein, (2) western blot analysis to establish i mmunoreactive equivalence between MON 87705- produced and the E. coli- produced reference standard protein using an anti-CP4 EPSPS antibody, (3) N-terminal sequence an alysis, (4) matrix-assisted laser desorption/ionization time-of-flight mass sp ectrometry (MALDI-TOF MS) to generate a tryptic peptide map of MON 87705-produced CP4 EPSPS, (5) CP4 EPSPS enzymatic activity analysis to demonstrate functi onal equivalence between MON 87705-produced

and the E. coli- produced reference standard protein, and (6) glycosyla tion analysis toMonsanto Company 09-SY-201U Page 87 of 471 establish equivalent gl ycosylation status betw een MON 87705-produced and E. coliproduced reference standard protein. The de tails of the materials, methods, and results are described in Appendix C while the conclusions are summarized below.

A comparison of the MON 87705-p roduced CP4 EPSPS to the E. coli- produced CP4 EPSPS reference standard protein confirmed the identity of the MON 87705- produced CP4 EPSPS protein and established the equivalence of the plant produced protein to the E. coli- produced CP4 EPSPS reference standa rd protein. The molecular weight of the MON 87705-produced and E. coli- produced CP4 EPSP S protein was estimated by SDS-PAGE. The SDS-PAGE dem onstrated that the proteins migrated identically, indicating that th e CP4 EPSPS proteins from both sources are equivalent in their molecular weight. The electrophoretic mobility and immunoreactive properties of the MON 87705-produced CP4 EPSPS protein were shown to be equivalent to those of

the E. coli- produced CP4 EPSPS prot ein reference standard by immunoblot. The Nterminus of the MON 87705 produced CP4 EPSPS protein was consistent with the predicted amino acid sequence translated fr om the CP4 EPSPS coding sequence, and the MALDI-TOF MS analysis yielded peptide mass es consistent with the expected peptide

masses from the translated CP4 EPSPS c oding sequence. The MON 87705- produced

and the E. coli- produced CP4 EPSPS protein reference standard were also found to be equivalent based on the functi onal activities and the lack of glycosylation. Taken together, these data provide a detailed charact erization of the CP4 EPSPS protein isolated

from MON 87705 and establishe d its equivalence to the E. coli -produced CP4 EPSPS reference standard protein. Furthermore, since CP 4 EPSPS proteins is olated from other Roundup Ready crops (Roundup Ready soyb ean, Roundup Ready 2 Yield soybean, Roundup Ready canola, Roundup Ready cotton and Roundup Ready sugar beet) have established equivalence to the E. coli- produced protein standa rd, by inference, the MON 87705-derived CP4 EPSPS protein is likely to possess equivale nt biochemical and physiological characteristics with the CP4 EPSPS proteins expressed in other Roundup Ready crops, all of which have been deregulated by USDA-APHIS.

VI.C. Expression Levels of CP4 EPSPS Protein in MON 87705

CP4 EPSPS protein levels in va rious tissues of MON 87705 that are relevant to the risk assessment were assessed by a validated enzy me-linked immunosorbent assay (ELISA).

Tissues of MON 87705 and c onventional control were co llected during the 2007/2008 growing season from five field sites in Chile (City, Pr ovince): Quilapilum, Chacabuco; Melipilla, Melipilla; Calera de Tango, Maipo; Rancagua, C achapoal; and San Fernando, Colchagua. These field sites were represen tative of soybean producing regions suitable for commercial production. At each site, th ree replicated plots of plants containing MON 87705, as well as a conventional soybean control, were plante d using a randomized complete block field design. Over-season leaf (OSL 1-4), root, forage, and mature seed tissues were collected from each replicated plot at all field sites. A description of tissues collected is provided below.Monsanto Company 09-SY-201U Page 88 of 471 Table VI-1. Tissues collected for MON 87705

Tissue Soybean development stage Days after planting (DAP)

OSL-1 V2-V3 31-35

OSL-2 V7 46-50

OSL-3 V10 62-66

OSL-4 V14 84-88 Forage R5-R6 101-106

Root R5-R6 101-106

Mature Seed R8 154-158

The CP4 EPSPS protein levels were determined in all seven ti ssue types described above. The results obtained from ELISA analysis are summarized in Table VI-2 and the details

of the materials and methods are describe d in Appendix D. In summary, the 2007/2008 Chilé expression study showed the CP4 EPSPS protein in MON 87705 was detected in all tissue types acro ss all five sites with a range from 40 – 1000 μg/g dwt. The levels of the CP4 EPSPS protein from the conventional control (A3525) were le ss than the assay limits of detection (LOD) or limit of quantitat ion LOQ in all tissue types. The mean CP4 EPSPS protein levels across the five sites we re highest in leaf (ranging from OSL-1 200 μg/g dwt to OSL-2 530 μg/g dwt), followed by root (120 μg/g dwt), seed (110 μg/g dwt) and forage (77 μg/g dwt).Monsanto Company 09-SY-201U Page 89 of 471 Table VI-2. Summary of CP4 EPSPS Protein Levels in Leaf, Seed, Root, and Forage

Tissues from MON 87705 Grown in 2007/2008 Chile Field Trials

Tissue Type 1 CP4 EPSPS protein

μ g/g fwt 2

(SD) 3 Range 4

μ g/g fwt 2 CP4 EPSPS protein

μ g/g dwt 5

(SD) 2 Range 4

μ g/g dwt 5 LOQ/LOD

μ g/g fwt 2

OSL-1 36 (14) 16-65 200 (72) 84-340 0.57/0.26

OSL-2 110 (51) 60-230 530 (230) 290-1000 0.57/0.26

OSL-3 51 (21) 11-84 220 (94) 47-350 0.57/0.26

OSL-4 51 (21) 27-94 210 (92) 110-410 0.57/0.26

Root 32 (5.3) 22-40 120 (24) 77-160 0.57/0.10

Forage 24 (6.4) 14-34 77 (24) 41-120 0.57/0.11

Mature

Seed 100 (39) 35-190 110 (44) 40-210 0.34/0.26

1 The OSL-1, OSL-2, OSL-3, OSL-4 samples were coll ected approximately at V2 – V3, V7, V10; and V14

stages, respectively. The forage and root were coll ected approximately at R5-R6 stage, and the mature seed was collected at R8 stage.

2 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight (fwt)

basis.

3 The means and standard deviations were calculated for each tissue type across all sites (n=15 for all tissues,

except OSL-2 where n=12 and OSL-3 where n=19).

4 Minimum and maximum values were determin ed for each tissue type across all sites.

5 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a dry weight (dwt)

basis. The dry weight values were calculated by dividing the μ g/g fwt by the dry weight conversion factors obtained from moisture analysis data.Monsanto Company 09-SY-201U Page 90 of 471 VI.D. Assessment of Potential Allergen icity of the CP4 EPSPS Protein from

MON 87705

According to guidelines adopt ed by the Codex Alimentarius Commission (Codex, 2003) for the allergy safety evaluation of novel pr oteins, the allergenic potential of a novel protein is assessed by compar ing the biochemical characteri stics of the novel protein to characteristics of known allergen s (Codex, 2003). A protein is not likely to be associated with allergenicity if: 1) the protein is from a nonallergenic source; 2) the protein represents only a very small portion of the total plant protein; 3) the protein does not

share structural similarities to known allergens based on the amino aci d sequence; and 4) the protein is rapidly digested in mammalian gastrointestinal systems. The CP4 EPSPS protein in MON 87705 has been assessed for its potential allergenicity according to these safety assessment guidelines.

The CP4 EPSPS protein is from Agrobacterium sp., strain CP4, an organism that is not a source of known allergens. Bioinformatics an alyses demonstrated that the CP4 EPSPS protein does not share immunologically releva nt amino acid sequence similarities with known allergens and, therefore, is highly unlikely to contain immunologically crossreactive allergenic ep itopes. Digestive fate experi ments conducted with the CP4 EPSPS protein demonstrated th at the full-length protein is rapidl y digested in simulated gastric fluid (SGF) and in simulated intestinal fluid (SIF) in vitro assays (Harrison et al., 1996). Finally, the CP4 EPSPS protein represents no more than 0.031% of the total protein in the seed of MON 87705, a relatively low abundan ce for this protein compared to the rest of the seed protein content. Taken together , these data support the conclusion that the CP4 EPSPS protein present in MON 87705 is not similar to known allergens and does not pose a significant allergenic risk to humans or animals.

VI.E. Safety Assessment Summa ry of CP4 EPSPS Protein

The U.S. Environmental Protection Agency (E PA) has established an exemption from the requirement of a tolerance for residues of CP4 EPSPS protein and th e genetic material necessary for its production in all plants (EPA, 1996). This exemption was based on a safety assessment that included rapid digest ion in simulated mammalian gastrointestinal fluids, lack of homology to t oxins and allergens, and lack of toxicity in an acute oral mouse gavage study. Since the MON 87705-pr oduced CP4 EPSPS protein is equivalent to the exempted CP4 EPSPS protein, a sim ilar conclusion can be reached that the MON 87705-produced CP4 EPSPS is safe fo r human and animal consumption. The comprehensive food and feed safety and nut ritional assessment of MON 87705 also is scheduled to be submitted to the FDA, which will include the following conclusions:

a) The donor organism, Agrobacterium species strain CP4 is not known for human or animal pathogenicity, and is not commonly allergenic. Agrobacterium sp. strain CP4 has been previously reviewed as a part of the safety assessment of the donor organism during Monsanto consulta tions with the FDA regarding Roundup Ready soybean (1994), Roundup Ready canola (1995), Roundup Ready cotton (1995), Roundup Ready Corn 2 (1996), Roundup Ready sugar beet (1998), Roundup Ready Flex cotton (2005), and R oundup Ready 2 Yield soybean (2007).Monsanto Company 09-SY-201U Page 91 of 471 b) EPSPS exerts its function in the shikimate pathway that is integral to aromatic amino acid biosynthesis in plants and microorganisms (Levin and Sprinson, 1964;

Steinrücken and Amrh ein, 1980). Therefore, this enzyme and its activity are found widely in food and feed derived from plant and microbial sources. Genes for numerous EPSPSs have been cloned (P adgette et al., 1996b) , and the catalytic domains of this group of proteins are c onserved. Bacterial EPSPSs have been well characterized with respect to their th ree dimensional X-ray crystal structures (Stallings et al., 1991) and detailed kinetic and chemi cal mechanisms (Anderson and Johnson, 1990).

c) The EPSPS from Agrobacterium sp. strain CP4 is highly tolerant to inhibition by glyphosate and has high catalytic effici ency, compared to most glyphosatetolerant EPSPSs (Barry et al., 1992; Padgett e et al., 1996b). The CP4 EPSPS protein thus represents one of many different EPSPSs found in nature, and the CP4 and native plant EPSPS enzymes are f unctionally equivalent except for their affinity to glyphosate. The CP4 EPSPS pr otein present in MON 87705 is similar to EPSPSs consumed in a variety of food and feed sources. CP4 EPSPS protein is homologous to EPSPSs naturally present in plants, including food crops (e.g., soybean and corn) and fungal and microbial food sources such as baker’s yeast ( Saccharomyces cerevisiae ), all of which have a history of safe human consumption (Harrison et al ., 1996; Padgette et al., 1996b). The si milarity of the CP4 EPSPS protein to EPSPSs in a variety of foods supports extensive human consumption of the family of EPSPS protei ns and the lack of health concerns. Furthermore, the ubiquitous presence of homologous EPSPS enzymes in food crops and common microorganisms establ ishes that EPSPS pr oteins, and their enzyme activity, pose no hazards for human and animal consumption.

d) The CP4 EPSPS protein does not share am ino acid sequence similarities with known allergens, gliadins, glutenins, or pr otein toxins which ha ve adverse effects to mammals. This has been demonstrated by extensive assessments with bioinformatic tools, such as the FASTA sequence alignment tool and eight-amino acid sliding window search. An amino acid sequence may be considered to have allergenic potential if it has an exact se quence identity of at least eight linearly contiguous amino acids with a potential allergen epitope (Metcalfe, 1996 Hileman et al., 2002). Using a sliding window of less than eight amino acids can produce matches containing significant uncer tainty depending on the length of the query sequence (Silvanovich et al., 2006) and are not useful to the allergy assessment process (Thomas et al., 2004).

e) The CP4 EPSPS protein is readily digestib le in simulated gastric and simulated intestinal fluids (Harrison et al., 1996). Rapid degradation of the full-length CP4 EPSPS protein in SGF and SIF makes it hi ghly unlikely for CP 4 EPSPS protein to be absorbed by epithelial cells of the small intestine in a biologically active form.

f) An acute toxicology study was conducted with a CP4 E PSPS protein (Harrison et al., 1996) that was shown to be physicochem ically and functionally equivalent toMonsanto Company 09-SY-201U Page 92 of 471 the CP4 EPSPS protein produced in MON 87705. Results indicate that the CP4 EPSPS protein did not cause a ny adverse effects in mice, with a No Observable Adverse Effect Level (NOAEL) of 572 mg/kg, the highe st dose level tested.

g) Potential human health risks from c onsumption of the CP4 EPSPS protein in

foods derived from MON 87705 were ev aluated by calculating a Margin of Exposure (MOE) between the acute mous e NOAEL for CP4 E PSPS protein and

95 th percentile “eater-only” estimates of acu te dietary exposure determined using the Dietary Exposure Eval uation Model (DEEM-FCID version 2.03, Exponent Inc.) and food consumption data fr om the 1994-1996 and 1998 USDA Continuing Survey of Food Intakes by Individuals (CSFII). The MOEs for acute dietary intake of the CP4 EPSPS protein were estimated to be 60,000 and 1,600 for the general population and non-nursi ng infants, respectively. These very large MOEs indicate that there is no meaningful risk to human health from dietary exposure to the CP4 EPSPS protein produced by MON 87705.

h) Potential health risks to animals from the presence of CP4 EPSPS protein in feed were evaluated by calculating an estimate of daily dietary intake (DDI). In the worst case scenario, the percentage of the CP4 EPSPS protein consumed from MON 87705 as part of the daily protein in take for a dairy cow is 0.0907% and for both the broiler and pig is less than 0.0325%.

Using the guidance provided by the FDA, a conc lusion of “no concern” is reached for the donor organism and the CP4 E PSPS protein. The food and feed products containing

MON 87705 or derived from M ON 87705 are as safe as soyb ean currently on the market for human and animal consumption.Monsanto Company 09-SY-201U Page 93 of 471 VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENT OF

MON 87705

Compositional comparisons between biotec hnology-derived and conventional crops represent an integral part of a nutritional and safety assessment. Compositional assessments are performed using the princi ples and analytes outlined in the OECD consensus documents for soybean compositi on (OECD, 2001). These principles are accepted globally and have been employed previously in assessments of soybean products derived thr ough biotechnology.

Compositional equivalence between biotech nology-derived and conventional crops provides an “equal or increased assurance of the safety of f oods derived from genetically modified plants” (OECD, 1998). The OECD consensus documents emphasize quantitative measurements of essential nutri ents, and known antinutrients and toxicants. This is based on the premise that such comprehensive and detailed analyses will most effectively discern any compositional ch anges that imply pot ential safety and

antinutritional concerns. Le vels of the components in seed and forage of the biotechnology-derived crop produc t are compared to: 1) co rresponding levels in a nonmodified comparator, typical ly the nontransgenic parental line grown under identical conditions, and 2) natural ranges generated fr om an in-study evalua tion of commercial varieties or from data published in the scientific literature.

MON 87705 was developed to generate soybean oil with lower levels of saturated fats (16:0 palmitic acid and 18:0 stearic acid) and higher levels of 18:1 oleic acid, with an

associated decrease in 18:2 linol eic acid, through suppression of FAD2 and FATB RNAs (Figure VII-1). MON 87705 contains the same major fatty acids that are found in conventional soybean, including 16:0 palmitic, 18:0 stearic, 18:1 oleic, 18:2 linoleic and 18:3 linolenic fatty acids. MON 87705 has a fatty acid profile that is comparable to other commercial high oleic vegetable oils (high oleic canola, high oleic safflower, high oleic sunflower), traditional oils, such as olive oil, that have a long-history of consumption in the diet, and canola oil that was granted GRAS status by the U.S. FDA. MON 87705

also contains the cp4 epsps gene encoding the CP4 EPSPS protein that is expressed throughout the plant conferring tolerance to glyphosate, the active ingredient in the Roundup family of agri cultural herbicides.

Compositional analyses were conducted to assess whether the nutrient and antinutrient levels in the seed and forage derived fr om MON 87705 are comparable to those in the conventional soybean control, A3525, wh ich has background genetics similar to MON 87705, but lacks the introduced traits. In addition, commercial conventional soybean varieties were included in the seed and forage composition analyses to establish a range of natural variability for each analyte, defined by a 99% tolerance interval. Statistically significant diffe rences were determined at the 5% level of significance (p<0.05) using establis hed statistical methods.

Seed and forage of MON 87705 and the conve ntional soybean control were harvested from soybean grown in three replicated plots, planted in a randomized complete block design, at each of five sites across Chile during the 2007- 2008 growing season: Quilapilun, Chacabuco Province (QUI); Melip illa, Melipilla Province (MEL); Calera de Tango, Maipo Province (CdT); Rancagua , Cachapoal Province (RAN); and SanMonsanto Company 09-SY-201U Page 94 of 471 Fernando, Colchagua Province (SFR). Sample s from all three replicates of MON 87705

and the control were collected from all th ree plots and analyzed . Four different commercial reference soybean varieties also were grown at each site for a total of 20 varieties. Samples from the commercial re ference varieties grown at each site were collected from all three plots. All replicates from 19 of 20 commercial conventional reference soybean varieties were analyzed ; however, one reference variety had all replicates damaged by an early frost and was excluded from the study. All MON 87705, control and reference soybean varieties were grown under normal agronomic field conditions for their respective geographic region s. Forage was collected at the R6 plant growth stage, and harvested soybean seed wa s collected at physiolo gical maturity. The seed and forage collected from MON 87705, th e conventional control, and the reference varieties were analyzed fo r compositional components.

In all, 67 analytical components were measur ed, 60 in seed and seven in forage. The analytes in forage included proximates (ash, fat, moisture , protein, and carbohydrates by calculation), acid detergent fiber (ADF), a nd neutral detergent fiber (NDF). Seed samples were analyzed for proximates, ADF, ND F, amino acids (18), fatty acids (26; C8- C24), trypsin inhibitors, phytic acid, lect in, isoflavones (daidzei n, glycitein, and genistein), vitamin E, raffinose, and stach yose. Materials and methods used for compositional analysis of the seed and fo rage of MON 87705, the conventional soybean control, and commercial conventional refe rence soybean varieties are presented in Appendix E.

The composition data then were statistically compared to that of the conventional soybean control to establish substantial equivalence. Of the measured components, 17 fatty acids in seed had more than 50% of the observations below the assay limit of quantitation (LOQ) and could not be statistically analyzed. Thus, statistical analyses were conducted for 50 components (43 in seed and seven in forage). The data set was examined for evidence of biologically relevant changes using a mixed model of variance. Six sets of statistical analys es were conducted, five based on the data from each of the replicated field sites (indivi dual-site) and the sixth anal ysis based on data from a combination of all five field sites (combine d-site). The statistical summaries of the combined-site analysis and the individual-site analyses, reported literature and the International Life Sciences Institute-Crop Composition Database (ILSI-CCD at http://www.cropcomposition.org) ranges for the analytical components present in seed are provided in Appendix E. The compositional data set was examined for evidence of statistically significant differences (p ≤ 0.05) between MON 87705 and the conventional soybean control. A summary of the significant differences observed between MON 87705 and the control are presented in Table VII-2.

Results of the comparisons indicate that except for the intended fatty acid changes, the composition of the seed and forage of MON 87705 is equivalent to that of the conventional soybean control A3525, in accord ance with OECD guidelines. Moreover, no new fatty acids beyond thos e presently found in soybea n were detected in MON 87705. Therefore, MON 87705 is regarded as safe and nutritious as conventional soybean for food and feed use. Further details of this assessment ar e provided in Section

VII-A.Monsanto Company 09-SY-201U Page 95 of 471 Figure VII-1. Schematic of the Soybean Fatty Acid Biosynthetic Pathway and Summary of Modified Fatty Acids in MON 87705

Panel A: Schematic of the soybean fatty acid biosynthetic pathway,

Panel B: MON 87705 soybean oil compared to commodity soybean oil and other vegetable oils

Indicates suppression of endogenous FATB and FAD2 in MON 87705 seeds

VII.A. Overall Assessment of the Compos ition of Forage and Seed from MON 87705 Compared to the Conventional Soybean Control

Based on the comprehensive assessment pr ocedures discussed above, MON 87705 is compositionally equivalent to conventional soybean except for the intended changes in fatty acid levels. Combined-site analysis of both forage and seed samples showed no statistically significant diffe rence (p>0.05) between MON 87705 and the control for 39 of 50 comparisons. Significant differences (p<0.05) between MON 87705 and the conventional soybean control were detected for 10 analytes in seed (arg inine, cystine, fat, 16:0 palmitic acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic acid, 20:0 arachidic acid, and 20:1 eicosenoi c acid) and one analyte (ash) in forage.

The compositional analyses confirmed that MON 87705 had the intended changes in the levels of four major soybean oil fatty acids (16:0 palmitic, 18:0 stearic, 18:1 oleic and 18:2 linoleic). For the remaining seven co mparisons where a significant difference (p<0.05) was detected, an analysis, in cluding magnitude of the differences,Monsanto Company 09-SY-201U Page 96 of 471 reproducibility across individual sites, and comparisons of mean analyte values to the 99% tolerance interval and literature values, i ndicated they were not materially different and were not biologically meaningf ully different from a food a nd feed safety perspective. Further assessment of the statistically si gnificant differences observed between MON 87705 and the conventional soybean contro l is provided in the following sections. Therefore, the compositional assessment of MON 87705 supports th e conclusion that, except for intended changes in seed fatty acid composition, seed and forage produced from MON 87705 are compositionally equivalent to those of conventional soybean.

VII.A.1 Intended Changes to Fatt y Acid Levels in MON 87705 Seed

As described previously, MON 87705 was devel oped to generate soybean oil with lower levels of saturated fats (16:0 palmitic acid and 18:0 stearic acid) and higher levels of 18:1 oleic acid, with an associ ated decrease in 18:2 linolei c acid, through suppression of FATB

and FAD2 RNAs (Figure VII-1). As expected, a ll of the intended changes in fatty acid levels were statistically significant in the co mbined-site analysis and are summarized in

the Table VII-1 below.Monsanto Company 09-SY-201U Page 97 of 471 Table VII-1. Summary of Intended Change s in Fatty Acid Levels for MON 87705
vs. the Conventional Soybean Control (A3525) in the Combin ed-Site Analysis

Fatty Acid (% total) MON 87705

Mean [Range] A3525

Mean [Range] Commercial

Tolerance

Interval 2

16:0 Palmitic 1 2.36 [2.25 - 2.44] 10.83 [10.51 – 11.08] [7.62, 12.55]

18:0 Stearic 1 3.31 [3.07 - 3.82] 4.50 [4.24 – 4.85] [2.87, 7.15]

18:1 Oleic 1 76.47 [73.13 - 79.17] 22.81 [21.41 – 25.08] [18.40, 30.22]

18:2 Linoleic 1 10.10 [7.85 - 12.42] 52.86 [51.68 – 53.89] [47.75, 56.46]

1 Significance level = <0.001

2 With 95% confidence, interval contains 99% of the values expresse d in the population of commercial soybean varieties. Negative limits were set to zero.

The results show that the level of the saturated 16:0 palmitic acid decreased from 10.83%

total FA in the conventional control to 2. 36% total FA in MON 87705, and the level of

the saturated 18:0 stearic acid decreased from 4.50% total FA in th e conventional control to 3.31% total FA in MON 87705. Thus, total saturated fat was decreased from approximately 15.3% total FA in the conve ntional control to 5.7% total FA in MON 87705. In addition, the mean level of 18:1 oleic acid increased from 22.81% total

FA in the conventional contro l to 76.47% total FA in MON 87705. These changes were

associated with a decrease in the mean leve l of 18:2 linoleic acid from 52.86% total FA

in the conventional control to 10.10% total FA in MON 87705. As expected, all intended changes in fatty acid levels in the seed of MON 87705 were st atistically significant in the combined-site analysis and consistently observed at all five of the individual sites. Thus, compositional analysis confirmed MON 877 05 had the intended fatty acid profile required for improved nutrition and soybean oil stability.

VII.A.2 Fatty Acid Levels in Soybean Seed

Of the 26 fatty acids analyzed in seed, 17 fatty acids had more than 50% of the observations below the assay limit of quantitation and, as a result, were excluded from the statistical analysis. Of the nine fatty acids that could be statistically analyzed, significant differences (p<0.05) were observed for seven fatty acids in the combined-site analysis (Table VII-2). Four of these diffe rences were due to the intended changes in fatty acids, as described in S ection VII.A.1. The three rema ining significant differencesMonsanto Company 09-SY-201U Page 98 of 471 in the combined-site analysis were for 18:3 linolenic, 20:0 arachidic and 20:1 eicosenoic acids. Given the intended changes in fatty acid metabolism, these additional differences in fatty acid levels were not unexpected. The biological relevance of these differences was assessed based on the magnitude of the difference, reproducibility across sites, and comparison of mean analyte values to the 99% tolerance interval for the population of commercial conventional soybean varieties grow n concurrently at the same field sites.

A combined-site statistical differen ce (p<0.05) between MON 87705 and the

conventional control was observed in the levels of 18:3 linolenic ac id. The decrease in linolenic acid is expected given that it is produced from 18:2 linoleic acid which was reduced by the suppression of the FAD2 gene. Examination of the reproducibility within

sites shows the levels of 18:3 linolenic acid were significantly lower than the soybean

control in four of five indi vidual-site analyses, with the absolute magnitude of the differences being small (<1.5% total FA; Appendix E). In add ition, all the mean levels of

18:3 linolenic acid in MON 87705 seed from the combined-site and individual-site analyses were well within the 99% tolerance interval, and therefore these differences are not considered biologically re levant compositional changes.

Combined-site statistical differences be tween MON 87705 and the conventional control

also were observed in levels of two minor fatty acids, 20:0 ar achidic acid, and 20:1

eicosenoic acid. The mean level of 20:0 arachidic acid in MON 87705 was significantly lower than in the conventional soybean cont rol in the combined-site analysis. An examination of the reproducibility within sites show ed that the levels of 20:0 arachidic acid were consistently lower than the soybean c ontrol in all five indi vidual-site analyses. However, the absolute magnitude of the differences was small (<0.063% total FA; Appendix E), all combined-sit e and individual-site means were well within the 99% tolerance interval, and therefore these differenc es are not considered biologically relevant compositional changes.

The mean level of 20:1 eicosenoic acid in MON 87705 was significantly higher than in the conventional soybean control in the combin e-site analysis. An examination of the reproducibility within sites showed that the levels of 20:1 eicosenoic acid were consistently higher than the soybean contro l in all five individual-site analyses. However, the absolute magnitude of these differences was small (<0.19% total FA; Appendix E). The combined-site mean for 20:1 eicosenoic (0.18% total FA) was slightly (0.09% total FA) outside the upper end (0.25% total FA) of the 99% tolerance interval but within the values reported in ILSI-CCD . In addition, 20:1 eicosenoic acid has a history of consumption in other commonly consumed vege table oils, such as canola (4.3% total FA), corn (0.6% total FA), mustar d seed (13.0% total FA), peanut (1.7% total FA), high oleic safflower (0 .5% total FA, and high oleic sunflower (0.5% total FA) (Codex, 2005). Therefore, the small change in the mean level of 20:1 eicosenoic acid in MON 87705 is not considered a biologically relevant compositional change.

These results lead to the conclusion that apar t from the intended changes in the levels of four fatty acids (16:0 palmitic, 18:0 stearic, 18:1 oleic and 18:2 linoleic), the seed from MON 87705 is compositionally equivalent to conventional soybean with regard to the levels of other fatty acids. The differences observed for 18:3 li nolenic, 20:0 arachidic, and 20:1 eicosenoic fatty acids were not une xpected, given the intended shift made in fatty acid metabolism (see Figure VII-1). Furt hermore, the mean levels of these fattyMonsanto Company 09-SY-201U Page 99 of 471 acids were within the 99% tolerance interval and/or the ILSI-CCD a nd literature values. Therefore, these differences are not consider ed biologically meaningf ul from a food and feed safety or nutritional perspective.

VII.A.3. Levels of Non-Fatty Acid Nutrients in Soybean Seed

In addition to fatty acids, soybean seed also was analyzed for the following 26 nutrients: proximates (5), ADF, NDF, amino acids (18), an d vitamin E. No statistically significant differences (p<0.05) were observed for 23 nu trient analytes. Th ree analytes were statistically different (p<0.05) between MON 87705 and the conventio nal control in the combined-site analysis: total fat, arginine and cystine (Table VII-2). The biological

relevance of these differences was assessed based on the magnitude of the difference, reproducibility across sites, and comparison of mean analyte values to the 99% tolerance interval for the population of commercial conventional soybean varieties grown concurrently at the same field sites. Mean analyte values we re further compared to ILSICCD and literature ranges.

The mean level of total fat was significan tly lower (p<0.05) in MON 87705 than the conventional soybean control in the combin ed-site analysis; however, the absolute magnitude of the mean difference was small (1.04% dw; Appendix E). There were no differences in total fat in any of the individual-site analyses. Furthermore, the mean level

of total fat in MON 87705 was well within th e 99% tolerance interv al. Therefore, the difference in total fat in MON 87705 compar ed to the control is not considered biologically meaningful.

The mean level of cystine was significan tly higher (p<0.05) in MON 87705 than the conventional soybean control in the combin ed-site analysis; however, the absolute magnitude of the mean difference was sma ll (0.022% dw; Appendix E). There were no differences in cystine levels in any of the individual-site anal yses. Furthermore, the mean level of cystine in MON 87705 was within the 99% tolerance interval. Therefore, the difference in cystine in MON 87705 compar ed to the control is not considered biologically meaningful.

The mean level of arginine was significan tly higher (p<0.05) in MON 87705 than the conventional soybean control in the combin ed-site analysis; however, the absolute magnitude of the mean difference was sm all (0.1% dw). Examination of the reproducibility within sites show s that the mean level of argi nine was significantly higher in only one of five i ndividual-site analyses; however, the absolute magnitude of the mean difference was small (0.18% dw; Appendix E) These differences are not biologically relevant changes in composition, given that th e mean levels of arginine in MON 87705 in the combined-site and individual-site analys es were all well within the 99% tolerance

interval.

These results lead to the conclusion that the seed from MON 87705 is compositionally equivalent to conventional soybean with regard to th e levels of nutrients. The differences observed for nutrients were limited in number, not consistently observed across sites, and

reflect the natural variation of conventional soybean. Furthermore, the mean levels of nutrient analytes were within the 99% tolerance interval and ILSI-CCD values.Monsanto Company 09-SY-201U Page 100 of 471 Therefore, these differences are not consider ed biologically meaningf ul from a food and feed safety or nutritional perspective.

VII.A.4. Naturally Occurring Anti-N utrient Levels in Soybean Seed

Soybean seed contains several well-describe d antinutritional factors according to OECD 

### ( 2001 

### ) , which include: trypsin inhibitors, lectin s, isoflavones (genistein, daidzein and glycitein), stachyose, ra ffinose, and phytic acid. Combined -site analysis of antinutrients showed no significant differences (p>0. 05) between MON 87705 and the conventional soybean control. Additional information is provided below to complete the discussion for the group of antinutrients.

Trypsin inhibitors are heat-labil e antinutrients that interfere with the digestion of proteins

and result in decreased animal growth (Liener, 1994). Lectin s are also heat labile, and can inhibit growth and cause death in animals if raw soybean is cons umed (Liener, 1994). Both trypsin inhibitors and lectins are inac tivated during processi ng of soybean protein products or soybean meal and, when proce ssed appropriately, the final edible soybean

fractions should contain minimal levels of these antinutri ents. No significant differences (p ≥ 0.05) were observed in trypsin inhi bitor levels between MON 87705 and the conventional soybean control in the combined-site or individual-site analyses.

There are three principl e isoflavones in soybean seed, na mely daidzein, genistein, and glycitein. Although they have been reported to possess bi ochemical activities, including estrogenic and anti-estrogenic effects, it is not universally accepted that the isoflavones are antinutrients because they have also b een reported to have be neficial antioxidant, anticarcinogenic and heart-heal thy hypocholesterolemic effect s (OECD, 2001). It is well

documented that isoflavone leve ls in soybean seed are high ly variable and are greatly influenced by many factors (OECD, 2001; Messina, 2001; Nelson et al., 2001). No significant differences (p ≥ 0.05) in isoflavone levels we re observed between MON 87705 and the conventional soybean control for the combined-site or individual-site analyses.

Stachyose and raffinose are low molecular weig ht carbohydrates presen t in soybean seed

that are considered to be antinutrients due to their consumption, whic h causes flatulence. No significant differences (p ≥ 0.05) in raffinose levels were observed between MON 87705 and the conventional soybean control in the combined-site or individual-site analyses. Stachyose levels showed no differences between MON 87705 and the conventional soybean control in the combined-site analysis, but were different at one site (Table VII-2). This difference is not consid ered biologically rele vant because it was observed only at one site and was not observed consistently across all sites.

Phytic acid present in soybean seed chel ates mineral nutrient s, including calcium, magnesium, potassium, iron and zinc, rende ring them biologically unavailable to monogastric animals consuming the seed (L iener, 2000). Unlike trypsin inhibitors, phytic acid is not heat labile, and remains stable through most soybean processing steps. No significant differences (p ≥ 0.05) in phytic aci d levels were observed between MON 87705 and the conventional soybean contro l for the combined-site or individualsite analyses.Monsanto Company 09-SY-201U Page 101 of 471 Based on the data and information presented above, it is concluded that the seed from MON 87705 is compositionally equivalent to conventional soybean with regard to the

levels of antinutrients.

VII.A.5. Proximate and Fiber Levels in Forage

Combined-site analysis of forage showed one significant differen ce (p<0.05) between MON 87705 and the conventional soybean control for ash. The biological relevance of

this difference was assessed based on the ma gnitude of the differe nce, reproducibility across sites, and comparison of mean analyte va lues to the 99% tolerance interval for the population of commercial conventional soybean varieties gr own concurrently at the same

field sites.

The mean level of ash wa s significantly higher (p<0 .05) in MON 87705 than the conventional soybean control; however, the absolute magnit ude of this difference was small (0.57% dw). There were no differen ces in ash in any of the individual-site analyses. Furthermore, the mean level of ash in MON 87705 was well within the 99%

tolerance interval. Theref ore, the difference in ash in MON 87705 compared to the control is not considered biologically meaningf ul. These results lead to the conclusion that the forage from MON 87705 is compositionally equivalent to that from conventional soybean.

VII.B. Compositional Equivalence of MON 87705 Seed and Forage to Conventional Soybean

Consistent with OECD guidelines for soyb ean composition (OECD, 2001) compositional analyses were conducted to assess whether le vels of nutrients, antinutrients, and key secondary metabolites in seed and forage derived from MON 87705 are comparable to those in the conventional soyb ean control, A3525, which has background genetics similar

to MON 87705 but lacks the introduced im proved fatty acid profile and glyphosate tolerance traits. Intended changes in the levels of the seed fatty acids 16:0 palmitic, 18:0 stearic, 18:1 oleic, and 18:2 lin oleic comprised four of the 11 significant differences in the combined-site analyses. For the remaining seven comparisons where a significant difference (p<0.05) was detected, an anal ysis, including magnitude of differences, reproducibility across indi vidual sites, and comparisons of m ean test analyte values to the 99% tolerance interval and pub lished values, indicates the di fferences are not materially

different and/or not bi ologically meaningful from a food and feed safety or nutritional perspective. Therefore, the compositio nal and nutritional assessment of MON 87705 supports the conclusion that, except for intende d changes in seed fatty acid composition, forage and seed produced from MON 87705 are compositionally equivalent to those of conventional soybean.Monsanto Company 09-SY-201UPage 102 of 471Table VII-2. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON 87705 vs. theConventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialToleranceInterval2Statistical Differences Observedin Combined-Site AnalysisForage Proximate (% DW)Ash8.758.186.990.020[7.39 - 10.11][6.78, 9.91]Seed Amino Acid (% DW)Arginine2.782.683.740.048[2.43 - 3.16][1.81, 3.62]Cystine0.610.593.660.043[0.57 - 0.64][0.49, 0.69]Seed Fatty Acid (% Total FA)16:0 Palmitic2.3610.83-78.18<0.001[2.25 - 2.44][7.62, 12.55]18:0 Stearic3.314.50-26.39<0.001[3.07 - 3.82][2.87, 7.15]18:1 Oleic76.4722.81235.20<0.001[73.13 - 79.17][18.40, 30.22]18:2 Linoleic10.1052.86-80.90<0.001[7.85 - 12.42][47.75, 56.46]Monsanto Company 09-SY-201UPage 103 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525)and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in Combined-Site AnalysisSeed Fatty Acid (% Total FA)18:3 Linolenic6.698.02-16.59<0.001[5.55 - 7.81][4.97, 9.93]20:0 Arachidic0.300.34-11.720.005[0.28 - 0.36][0.22, 0.53]20:1 Eicosenoic0.340.1979.85<0.001[0.27 - 0.40][0.13, 0.25]Seed Proximate (% DW)Total Fat18.2919.33-5.38<0.001[16.55 - 19.50][15.35, 25.95]Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)16:0 Palmitic Site CdT2.3110.80-78.62<0.001[2.29 - 2.32][7.62, 12.55]16:0 Palmitic Site MEL2.3910.83-77.92<0.001[2.35 - 2.42]16:0 Palmitic Site QUI2.3010.56-78.240.005[2.25 - 2.37]Monsanto Company 09-SY-201UPage 104 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)16:0 Palmitic Site RAN2.4010.96-78.12<0.001[2.39 - 2.40][7.62, 12.55]16:0 Palmitic Site SFR2.4211.00-78.00<0.001[2.40 - 2.44]18:0 Stearic Site CdT3.174.58-30.88<0.001[3.09 - 3.23][2.87, 7.15]18:0 Stearic Site MEL3.334.39-24.060.018[3.20 - 3.47]18:0 Stearic Site QUI3.514.82-27.200.004[3.15 - 3.82]18:0 Stearic Site RAN3.344.50-25.730.001[3.28 - 3.41]18:0 Stearic Site SFR3.224.31-25.260.001[3.07 - 3.41]Monsanto Company 09-SY-201UPage 105 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:1 Oleic Site CdT76.4423.02232.08<0.001[76.35 - 76.60][18.40, 30.22]18:1 Oleic Site MEL76.1022.31241.09<0.001[75.68 - 76.33]18:1 Oleic Site QUI78.6124.95215.050.003[77.70 - 79.17]18:1 Oleic Site RAN74.6921.53246.87<0.001[73.13 - 75.98]18:1 Oleic Site SFR76.4922.42241.12<0.001[75.33 - 77.21]18:2 Linoleic Site CdT10.0952.43-80.75<0.001[9.94 - 10.22][47.75, 56.46]18:2 Linoleic Site MEL10.5053.48-80.38<0.001[10.16 - 10.92]Monsanto Company 09-SY-201UPage 106 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:2 Linoleic Site QUI8.7551.70-83.070.006[7.85 - 10.02][47.75, 56.46]18:2 Linoleic Site RAN11.3253.73-78.92<0.001[10.37 - 12.42]18:2 Linoleic Site SFR9.8252.84-81.42<0.001[9.33 - 10.55]18:3 Linolenic Site CdT6.908.15-15.320.001[6.85 - 6.94][4.97, 9.93]18:3 Linolenic Site MEL6.588.00-17.720.002[6.53 - 6.65]18:3 Linolenic Site QUI5.647.02-19.690.029[5.55 - 5.71]18:3 Linolenic Site SFR6.988.49-17.720.009[6.79 - 7.26]Monsanto Company 09-SY-201UPage 107 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)20:0 Arachidic Site CdT0.290.35-18.100.016[0.28 - 0.29][0.22, 0.53]20:0 Arachidic Site MEL0.300.34-11.860.026[0.29 - 0.30]20:0 Arachidic Site QUI0.330.36-8.840.041[0.30 - 0.36]20:0 Arachidic Site RAN0.280.33-13.080.014[0.28 - 0.29]20:0 Arachidic Site SFR0.290.32-8.180.006[0.29 - 0.29]20:1 Eicosenoic Site CdT0.360.2176.81<0.001[0.36 - 0.38][0.13, 0.25]20:1 Eicosenoic Site MEL0.350.2070.850.001[0.34 - 0.36]Monsanto Company 09-SY-201UPage 108 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)20:1 Eicosenoic Site QUI0.380.2089.530.049[0.37 - 0.40][0.13, 0.25]20:1 Eicosenoic Site RAN0.290.1682.180.003[0.27 - 0.31]20:1 Eicosenoic Site SFR0.330.1880.720.005[0.32 - 0.35]Seed Fiber (% DW)Acid Detergent Fiber Site CdT18.2316.2712.100.049[17.57 - 18.58][12.71, 19.29]Acid Detergent Fiber Site RAN16.3213.9417.070.002[15.71 - 16.78]Statistical Differences Observed in One SiteForage Proximate (% DW)Carbohydrates Site RAN69.7772.09-3.220.027[68.94 - 71.06][64.45, 80.50]Total Fat Site MEL5.796.76-14.290.030[5.37 - 6.57][0, 9.74]Monsanto Company 09-SY-201UPage 109 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in One SiteSeed Amino Acid (% DW)Alanine Site SFR1.511.444.620.024[1.49 - 1.54][1.25, 1.92]Arginine Site SFR2.522.347.560.047[2.43 - 2.64][1.81, 3.62]Aspartic Acid Site SFR3.763.565.480.009[3.67 - 3.88][3.02, 5.11]Glutamic Acid Site SFR5.905.536.620.008[5.72 - 6.12][4.42, 8.48]Histidine Site SFR0.900.855.900.018[0.88 - 0.94][0.74, 1.16]Leucine Site SFR2.542.415.120.014[2.47 - 2.61][2.06, 3.41]Lysine Site SFR2.252.135.320.007[2.19 - 2.30][1.87, 2.81]Monsanto Company 09-SY-201UPage 110 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in One SiteSeed Amino Acid (% DW)Phenylalanine Site SFR1.681.604.830.019[1.64 - 1.73][1.35, 2.31]Proline Site SFR1.621.555.020.021[1.59 - 1.66][1.29, 2.21]Tyrosine Site SFR1.181.125.720.042[1.17 - 1.20][0.99, 1.49]Seed Fatty Acid (% Total FA)24:0 Lignoceric Site CdT0.150.15-3.240.008[0.14 - 0.15][0.030, 0.26]Seed Fiber (% DW)Neutral Detergent Fiber Site CdT21.0417.9916.970.009[20.47 - 22.18][12.07, 21.51]Seed Proximate (% DW)Carbohydrates Site CdT41.8240.054.400.016[41.62 - 42.00][30.78, 45.86]Seed Vitamin (mg/100g DW)Vitamin E Site MEL3.263.83-15.050.005[3.15 - 3.45][0, 7.36]Monsanto Company 09-SY-201UPage 111 of 471Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison ofSoybean Component Levels for MON 87705vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMeanDifference(% of A3525)Significance(p-Value)MON 87705RangeCommercialTolerance Interval2Statistical Differences Observed in One SiteSeed Antinutrient (% DW)Stachyose Site CdT3.763.1021.270.046[3.55 - 4.16][1.96, 4.41]1DW = dry weight; FA = fatty acid.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were setto zero.Monsanto Company 09-SY-201U Page 112 of 471 VII.C. Compositional Comparison of Pro cessed Fractions from Soybean Seed of

MON 87705 and the Conventional Control

To prepare soybean processed fractions, seed samples were collected from field trials

conducted with MON 87705 and the conventiona l soybean control at two field sites (Jefferson County, IA and C linton County, IL) in the U.S. during the 2007 growing season. In addition, 12 commercial conventio nal soybean varieties were grown at three field sites in the U.S. and processed to determine a 99% tolerance interval for each component analyzed. The seed samples were processed into defa tted toasted soybean meal (TD soybean meal); refined, bleached , and deodorized soybean oil (RBD oil); protein isolate; and crude le cithin fractions. The proce ssed fractions were analyzed according to the principles outlined in the OECD consensus document for soybean composition (OECD, 2001). The TD soybe an meal was analyzed for proximates (moisture, protein, fat, as h, and carbohydrates by calcula tion), ADF, NDF, amino acids, trypsin inhibitors and phytic acid. The RBD oil was analyzed for fatty acids and vitamin E ( α -tocopherol). The protei n isolate fraction was analyzed for amino acids and

moisture. The crude lecithin fract ion was analyzed for phosphatides ( α -phosphatidic acid, α -phosphatidylcholine, α -phosphatidylethanolamine, and α -phosphatidylinositiol). Compositional analyses were conducted to assess whether the processed fractions from MON 87705 are comparable to those of the conventional soybean control, A3525, which has background genetics similar to MON 87705 , but lacks the introduced improved fatty acid profile and glyphosate tole rance traits. Statistically significant differences were

determined at the 5% level of significance (p<0.05) using es tablished statistical methods. The statistical analysis compared MON 87705 and the conventional control across the two sites (combined-site). Statistical summary of the composition of each processed fraction and summary of the significant di fferences observed between the processed fractions prepared from the seed of MON 87705 and the conventional control are included in Appendix E.

Results show that there were no statistically significant differences (p>0.05) between

MON 87705 and the conventional control for co mponents measured in the protein isolate fraction or phosphatides of crude lecithin . Comparison of the composition of TD soybean meal processed from MON 87705 and the conventional soybean control showed no differences (p>0.05) for 21 of the 27 comp onents analyzed. Significant differences (p<0.05) were observed for si x components of the TD soybean meal: alanine, glycine, isoleucine, lysine, valine, and NDF. The absolute magnitude of the differences was small (<1.8% dw) and the MON 87705 mean values fe ll within the 99% tole rance interval for the conventional soybean varieties and also within the range of published values for conventional soybean. The low levels of residu al oil (0.78% dw, as total fat) present in the TD soybean meal from MON 87705 also ar e expected to reflect the intended changes in fatty acid levels observed in seed.

As expected, and consistent with the resu lts obtained for seed fatty acid levels, the intended fatty acid changes (16:0 palmitic, 18:0 palmitic, 18:1 oleic and 18:2 linoleic) also were observed in RBD oil. In addition, six fatty acids were detected in RBD oil that were not detected in seed: 14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric (heptadecanoic) acid, 17:1 9c heptadecenoic acid, 18:2 other trans isomer fatty acidsMonsanto Company 09-SY-201U Page 113 of 471 (excluding 9t,12t linolelaidic), and 18:2 6c,9 c, octadecadienoic acid. As observed in seed, levels of several less abundant fatty ac ids were significantly different (p<0.05)

between the RBD oil from MON 87705 and the conventional control. Differences were observed for 14:0 myristic acid, 16:1 palmito leic acid, 17:0 marg aric (heptadecanoic) acid, 20:0 arachidic, 20:1 eicosenoic and 22:0 behenic acids. However, the absolute magnitude of the differences was small (<0.15% total FA), and the MON 87705 mean

values fell within the 99% tolerance interval s for the reference varieties and/or within published ranges for conven tional soybean oil (Codex, 2005; Appendix E).

A significant increase (p<0.05) in the level of the minor fatty acid 17 :1 9c heptadecenoic acid was observed in MON 87705 compared to conve ntional control RBD oil. This is not unexpected, given the intended shift in fatty acid levels in MON 87705. The mean level of 17:1 9c heptadecenoic acid in MON 87705 (0 .12% total FA) was out side the range of

values obtained for the RBD oil from comm ercial references. However, 17:1 9c heptadecenoic acid is present at similar or highe r levels in a variety of oils (canola, corn, peanut, high oleic safflower, and high oleic sunflower; C odex, 2005) and foods (tofu, ground beef, and soft-spread margarine; US DA-ARS, 2007). Therefore, there are no adverse food and feed safety or nutrition eff ects associated with the levels of 17:1 9c heptadecenoic acid observed in MON 87705 s oybean oil. The remaining minor fatty

acids 18:2 other trans (excluding linolelaidic) and 18:2 6c,9c, octadecadienoic acid, not

detected in seed and are be lieved to arise from the s pontaneous isomerization of unsaturated fatty acids during th e oil refining process. Levels of these fatty acids were significantly lower (p<0.05) in MON 87705 compared to cont rol RBD oil and thus, these

differences were not consider ed biologically relevant from a food and feed safety or nutritional perspective (C hardigny et al., 1996).

Therefore, this supports the conclusion that, except for the intended changes in fatty acid composition, minor differences in the levels of less abundant fatty acids and occurrence of low levels of minor fatty acids due to s pontaneous isomerisation during the o il refining process, the processed fractions pr oduced from MON 87705 are compositionally equivalent to those of conventional soybean.

VII.D Safety and Nutritional Assessmen t of the Intended Changes in MON 87705

MON 87705 was developed to generate soybean oil with decreased levels of saturated fats (16:0 palmitic acid and 18:0 stearic acid) and increased levels of 18:1 oleic acid, with

an associated decrease in 18:2 linoleic acid. Replacement of conventional soybean oil with MON 87705 soybean oil under the proposed f ood uses results in changes in the fatty acid composition in the U.S. diet that l ead to higher oleic acid intake,and lower consumption of saturated fats (i.e., 16:0 pa lmitic and 18:0 linoleic acid) with no impact

on total fat intake. This assessment assumes all of the targeted oil components of the foods proposed for replacement that are co nsumed in the U.S. are replaced with MON 87705 soybean oil. Therefore, the resu lts presented in this petition represent a theoretical maximal effect of MON 87705 soybean oil on fatty acid composition of the diet. The nutritional impact from the us e of MON 87705 soybean oil in targeted foods under the intended conditions of use is estimated to result in changes in fatty acid consumption that are within current dietary gu idelines for fatty acid intake (Lichtenstein et al., 2006; USDA-ERS 2005; WHO/FAO, 2 003). A discussion of the safety andMonsanto Company 09-SY-201U Page 114 of 471 nutritional impact re sulting from the intended changes in MON 87705 is included in Appendix M.

VII.E Safety and Nutrition Assessment Conclusion

In conclusion, except for the intended cha nges in fatty acid levels, the compositional equivalence of MON 87705 seed, forage, a nd processed fractions to conventional soybean has been demonstrated in accordance with OECD guidelines. In addition, the nutritional impact from the use of MON 87705 soybean oil in targeted foods under the

intended conditions of use is estimated to resu lt in changes in fatty acid consumption that are within current dietary guidelines for fa tty acid intake. Th erefore, MON 87705 is regarded to be as safe an d nutritious as conventional s oybean for food and feed use.Monsanto Company 09-SY-201U Page 115 of 471 VIII. PHENOTYPIC, AGRONO MIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides an eval uation of the phenotypic and ag ronomic characteristics and environmental interactions of MON 87705 comp ared to the conven tional A3525 control, a conventional soybean variet y that has background genetics similar to MON 87705 but does not possess the improved fatty acid pr ofile and glyphosate tolerance trait. Phenotypic and agronomic characteristic s of MON 87705 were evaluated in a comparative manner to assess plant pest po tential (OECD, 1993). In the phenotypic, agronomic, and environmental interactions assessment of MON 87705, data were collected to evaluate specific aspects of altered plant pest potential based on requirements of USDA-APHIS set forth at 7 CFR § 340.6. The MON 87705 plant characterization and

environmental interactions data cover six general categories: 1) germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive grow th (including pollen characteristics); 4) seed retention on th e plant and lodging; 5) plant-symbiont associations; and 6) plant interactions with insect, disease, and abiotic stressors. An

overview of the characteristics assess ed is presented in Table VIII-1.

Results from the phenotypic and agronomic assessments indicate that MON 87705 does not possess characteristics that would confer a plant pest risk or si gnificant environmental impact compared to conventional soybean. Data on environmental interactions also

indicate that MON 87705 does not confer any increased suscep tibility or tolerance to specific diseases, insects, or abiotic stressors.

VIII.A. Characteristics Measured for Assessment

The phenotypic, agronomic, and environmental interactions data were evaluated from a basis of familiarity (OECD, 1993) and were comprised of a combination of field, greenhouse, and laboratory studies conducted by scientists who are familiar with the production and evaluation of soybean. In each of these assessments, MON 87705 was compared to an appropriate conventional cont rol that had a genetic background similar to MON 87705 but did not possess th e improved fatty acid profile and glyphosate tolerance trait. In addition, multiple commercial s oybean varieties (see Appendix F and Tables F1, G-1, and I-1) were included to provide a range of comparative values that are representative of existing commercial soyb ean varieties for each measured phenotypic, agronomic, and environmental interaction ch aracteristic. Data collected from the commercial reference varieties reflect a rang e of selection and breeding for desirable characteristics and therefore can provide contex t for interpreting experimental results.Monsanto Company 09-SY-201U Page 116 of 471 Table VIII-1. Phenotypic, Agronomic and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials or Greenhouse Studies

Data Category Characteristics

measured Evaluation timing 1 Evaluation description (measurement endpoints)

Germination , dormancy,

and

emergence Normal germinated Day 5 and 8 (20/30°C) % of seed producing seedlings exhibiting normal developmental characteristics Abnormal germinated Day 8 (20/30°C) % of seed that could not be classified as normal germinated Germinated Day 5, 8, and 13 (10, 20, 30, 10/20 and 10/30°C) % of seed that had germinated normally and abnormally Dead Day 5 and 8 (10, 20, 30, 10/20, 10/30, and 20/30°C); Day 13 (10, 20, 30, 10/20 and 10/30°C) % of seeds that had visibly deteriorated

and had become soft to the touch

Hard viable and

nonviable Day 8 (20/30°C); Day 13 (10, 20, 30, 10/20 and 10/30°C) % of seeds that did not imbibe water and

remained hard to the touch

Firm swollen viable

and nonviable Day 8 (20/30°C); Day 13 (10, 20, 30, 10/20 and 10/30°C) % of seeds that imbibed water

Early stand count V2 - V4 Number of emerged plants in two rows,

standardized to 20 ft rows Final stand count Maturity, R8 Number of plants in two rows,

standardized to 20 ft rows

Vegetative growth Seedling vigor V2 - V4 Rated on a 1-9 scale, where 1-3 = excellent, 4-6 = average, and 7-9 = poor vigor Growth stage

assessment Every two-three weeks, V2-

R8 Average soybean plant growth stage per plot Flower color Flowering, R1-R2 Color of flowers: purple, white, or mixed Plant pubescence Maturity, R8 Pubescence on plants in each plot categorized as hairy, hairless, or mixed Plant height Maturity, R8 Distance from the soil surface to the uppermost node on the main stem of five representative plants per plot

Reproductiv e growth Days to 50% flowering Flowering, R1-R2 Calendar day number when approximately 50% of the plants in each plot were flowering Pollen viability Flowering, R1-R2 Viable and nonviable pollen based on pollen grain staining characteristics Pollen morphology Flowering, R1-R2 Diameter of viable pollen grains

Seed moisture Harvest Percent moisture content of harvested seed 100 seed weight (g) Harvest Mass of 100 harvested seeds Test weight (lb/bu) Harvest Mass of a bushel of harvested seed Yield (bu/ac) Harvest Bushels of harvested seed per acre, adjusted to 13% moisture

Seed

retention and lodging Lodging Maturity, R8 Rated on 0- 9 scale, where 0 = completely erect and 9 = completely flat or lodged

Pod shattering Maturity, R8 Rated on 0-9 scale, where 0 = no shattering and 9 = completely shattered

Plantsymbiont

interactions Biomass 6 weeks after emergence in greenhouse Nodule, root, and shoot dry weight

Nodule number 6 weeks after emergence in greenhouse Nodule number

Total nitrogen 6 weeks after emergence in greenhouse Shoot total nitrogen (% and g/plant)Monsanto Company 09-SY-201U Page 117 of 471 Table VIII-1 (continued). Phenotypic, Agronomic and Environmental

Interaction Characteristics Evaluated in U.S. Field Trials or Greenhouse Studies

Data Category Characteristics

measured Evaluation timing 1 Evaluation description (measurement endpoints)

Plant

interactions Plant response to

abiotic stressors and disease damage Four times per growing

season Qualitative assessment of each plot, with rating on a 0-9 scale, where 0 = no symptoms and 9 = severe symptoms Arthropod damage Four times during growing

season Qualitative assessment of each plot, with rating on a 0-5 scale, where 0 = no symptoms and 5 = severe symptoms Arthropod abundance Three times during growing

season Quantitative assessment of pest and beneficial arthropods

1 Soybean plant growth stages were determined usin g descriptions and guidelines outlined in Soybean Growth and Development (Pedersen, 2004).

VIII.B. Interpretation of Phenotypic and Environmental Interaction Data

Plant pest risk assessments for biotechnology- derived crops are, by standard, comparative assessments. Familiarity provides a basis from which the potential environmental impact of a biotechnology-derived plant can be evaluate d. The concept of familiarity is based on the fact that the biotechnology-derived plan t is developed from a well-characterized conventional plant variety. Familiarity considers the biology of the crop, the introduced trait, the receiving environment and the inter action of these factors, and provides a basis for comparative environmental risk assessm ent between a biotechnology-derived plant and its conventional counterpart.

Expert knowledge and experience with conve ntionally bred soybean was the basis for selecting appropriate endpoint s and estimating the range of responses that would be considered typical for soybean. As su ch, assessment of phenotypic and agronomic

characteristics and environmental intera ctions was essential to compare the biotechnology-derived plant to the conven tional counterpart. An overview of the characteristics assessed is presented in Table VIII-1. A subset of the data relating to

well-understood weediness criteria (e.g., seed dormancy, pre-harvest seed loss characteristics, and lodging) was used to assess whether there was an increase in weediness potential, an element of APHIS’s plant pest determination. Based on all of the data collected, an assessment was made whether the biotechnology-deri ved plant is likely to pose an increased plant pest risk compared to the conventional counterpart.

Experienced scientists familiar with each experimental design and evaluation criteria

were involved in all components of data collection, summ arization, and an alysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence with the crop, and the experiment was carefully monitored. Prior to analysis, the overa ll dataset was evaluated for evidence of biologically relevant changes, and for possible evidence of an unexpected plant response. These scientists did not identify any unexpected observations or issues in the course of

these evaluations. Data were then su bjected to statistical analysis.Monsanto Company 09-SY-201U Page 118 of 471 VIII.C. Interpretation of Detected Differences Criteria

Comparative plant characteriz ation data between a biot echnology-derived crop and the control are interpreted in the c ontext of contributions to increased plant pest potential as assessed by APHIS. Under the framework of familiarity, characteristics for which no differences are detected suppor t a conclusion of no increase d plant pest potential of the biotechnology-derived crop comp ared to the conventional cr op. Characteristics for

which differences are detected are considered in a step-wise method (Figure VIII-1 or a similar method). All detected differences fo r a characteristic ar e considered in the

context of whether or not th e difference would increase the plant pest potential of the biotechnology-derived crop. Ultimately, a weight of evidence approach considering all

characteristics and studies was used for the overall risk assessment of differences and their significance. In detail, Figure VIII-1 illustrates the stepwise assessment process employed:

Step 1

Note: A “no” answer at any step indicates that th e characteristic does not cont ribute to a biological or ecological change for the crop in terms of plant pest potential and subsequent steps are not considered. If the answer is “yes” or uncertain th e subsequent step is considered.

Figure VIII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods Hazard identification and

risk assessment on

difference Outside variation for crop? No

Yes

No

Yes Adverse in terms of plant pest potential? Yes Outside variation of study No Not adverse; the direction or magnitude of the

detected difference in the

measured characteristic

does not contribute to a biological or ecological change for the crop in

terms of plant pest potential Yes Statistical differences detected in combined-site analysis? No Differences detected in the combined-site and individual site analyses are evaluated*

Step 2

Step 3

Step 4 The measured

characteristic does not contribute to a biological or ecological change for the crop in terms of plant pest potential

Step 5

Step 6

*See text for interpretation of differences detected in the individual site analysisMonsanto Company 09-SY-201U Page 119 of 471 ▪ Steps 1 and 2. Evaluate Detected Statistical Differences. Combined-site and

individual-site statistical analyses are conducted and evaluated on each measured

characteristic. Differences detected in the individual-site analysis must be observed in the combined site analysis to be consider ed further for plant pest potential. Any

difference detected in the combined-s ite analysis is further assessed.

▪ Step 3. Evaluate Differences Relative to Reference Range. If a difference is detected in the combined-site analysis across multiple environments, then the test substance mean

value is assessed relative to the reference substances.

▪ Step 4. Evaluate Differences in the Context of the Crop. If the test substance mean is

outside the variation of the re ference substances (e.g., refere nce range), the test substance

mean is considered in the context of known values common for the crop.

▪ Step 5. Plant Pest Potential. If the test substance mean is outside the range of values common for the crop, the detected difference is then assessed for whether or not it is

adverse in terms of plant pest potential.

▪ Step 6. Conduct Risk Assessment on Identified Hazard. If an adverse effect (hazard) is identified, risk assessment on the difference is conducted. The risk assessment

considers contributions to enha nced plant pest potential of the crop itself, the impact of

differences detected in other measured charac teristics, and potential for and effects of trait transfer to feral populations of the crop or to a sexually co mpatible species.

VIII.D. Phenotypic, Agronomic and Envi ronmental Interactions Characteristics

As a significant part of the evaluation of MON 87705, plant phenotypic and agronomic characteristics including s eed dormancy and germinati on, phenotypic, agronomic and environmental interactions, pollen characteri stics, and symbiont interactions were evaluated. The phenotypic, agronomic, and environmental interaction evaluations are based on replicated laboratory, greenhouse, and/or multi-site field trials and experiments. In evaluating the phenotypic and agronomic characteristics of MON 87705, data were collected that address specific environmental risks regarding plant pe st potential based on

the considerations of USDA-APHIS.

VIII.D.1. Seed Dormancy and Germination Characteristic

APHIS considers the potential for weediness to constitute a plant pest factor (CFR § 340.6). Seed germination and dormancy mechanisms vary with species and their genetic basis tends to be complex. Seed dormancy (e.g ., hard seed) is an im portant characteristic that is often associated with plants th at are considered as weeds (Anderson, 1996; Lingenfelter and Hartwig, 2003), where in s oybean it is not uncommon to observe low levels of hard seed (Mullin and Xu, 2001; Potts et al., 1978). Standardized germination assays are available and routinely used to measure the germination characteristics of soybean seed. The Association of Official S eed Analysts, an internationally recognized seed testing organization, recomm ends a temperature range of 20/30 ° C as optimal for germination of soybean (AOSA, 2007).

Comparative assessments of seed dormanc y and germination characteristics were conducted on MON 87705 and A3525, where A 3525 served as a comparable controlMonsanto Company 09-SY-201U Page 120 of 471 because it has background genetics simila r to MON 87705 but does not possess the improved fatty acid profile and glyphosate tolerance tra it. In addition, eight commercially available soybean varieties were included as references to provide baseline values common to soybean. The seed lots for MON 87705, the conventional soybean

control and reference varie ties were produced during 2007 in Iowa (IA), Indiana (IN), and Missouri (MO), geographic areas whic h represent environmentally relevant conditions for soybean production for this product. In addition to the AOSA recommended temperature range of 20/30 °C, s eed was tested at five other temperature regimes of 10, 20, 30, 10/20, and 10/30 ° C to assess seed germination properties. The details of the materials, experimental met hods, and germination data from all individual production sites are presen ted in Appendix F.

No statistically significant differences were detected between MON 87705 and the control for percent viable hard seed in any temperature regime with the exception of the

20 ° C temperature regime (Table VIII-2). At 20 ° C, MON 87705 had lower percent

viable hard seed than the cont rol (0.0 vs. 0.3%). The mean value for percent viable hard

seed of MON 87705 was within the reference ra nge (0.0 – 0.3%). Thus, the statistical difference detected for percent hard seed is unlikely to be biologically meaningful in terms of increased weed potential of M ON 87705 compared to conventional soybean. Furthermore, a decrease in hard seed would not contribute to in creased weediness of soybean.

No statistically significant differences were detected between MON 87705 and the control for percent viab le firm swollen seed in any temp erature regime (Table VIII-2). Within some temperature regimes, it was not possible to conduct an analysis of variance for percent viable firm swollen seed because there was no variability present in the data. For these data, the values for MON 87705 and the control were a ll zero, indicating no biological differences. Additiona lly, no statistically significant differences were detected between MON 87705 and the control for percent germinated, viable hard, dead, or viable
firm swollen seed in the 10, 10/20, or 10/30 ° C temperature regimes.

Three other statistically si gnificant differences were detected between MON 87705 and the control in the combined-site analysis (Table VIII-2). MON 87705 had lower percent germinated seed than the control at 30 ° C (92.8 vs. 95.5%), and ha d higher percent dead

seed than the control at 30 ° C (7.3 vs. 4.5%) and 20/30 oC (2.7 vs. 1.3%). Lower percent germinated seed and higher percent dead seed would not contribute to increased weediness. Furthermore, all values were well within the recommended standards for certified soybean seed (AOSCA, 2009b).

The biological characteristics evaluated in this study were used to characterize MON 87705 in the context of plant pest ri sk assessment. Based on the dormancy and germination characteristics assessed, the results of this study, particularly the lack of increased hard seed, demonstrate there were no changes indicative of increased weed potential of MON 87705 relativ e to conventional soybean.Monsanto Company 09-SY-201U Page 121 of 471 Table VIII-2. Germination Charac teristics of MON 87705 and A3525

Temp. Regime 1 Category Mean % (S.E.) 2

MON 87705 A3525 Reference Range 3

10 °C Germinated 97.7 (0.7) 98.4 (0.4) 98.2 – 99.5

Viable Hard 0.1 (0.1) 0.3 (0.1) 0.0 – 0.8

Dead 2.1 (0.8) 1.1 (0.4) 0.3 – 1.3

Viable Firm-Swollen 0.2 (0.1) 0.3 (0.1) 0.0 – 0.5

20 °C Germinated 99.1 (0.5) 98.8 (0.3) 96.9 – 99.8

Viable Hard 0.0 (0.0) * 0.3 (0.1) 0.0 – 0.3

Dead 0.9 (0.5) 0.8 (0.3) 0.3 – 3.1

Viable Firm-Swollen 0.0 (0.0)† 0.0 (0.0) 0.0 – 0.0

30 °C Germinated 92.8 (2.4)* 95.5 (0.6) 94.4 – 99.5

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3

Dead 7.3 (2.4) * 4.5 (0.6) 0.3 – 5.4

Viable Firm-Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0

10/20 °C Germinated 99.3 (0.2) 99.6 (0.2) 98.4 – 100.0

Viable Hard 0.1 (0.1) 0.1 (0.1) 0.0 – 0.3

Dead 0.6 (0.1) 0.3 (0.2) 0.0 – 1.6

Viable Firm-Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0

10/30 °C Germinated 98.0 (0.7) 98.8 (0.4) 98.0 – 99.5

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3

Dead 2.0 (0.7) 1.3 (0.4) 0.5 – 1.8

Viable Firm-Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0

20/30 °C Normal Germinated 92.4 (1.0) 93.6 (0.7) 57.0 – 98.5
(AOSA) Abnormal Germinated 4.9 (0.7) 5.0 (0.7) 1.3 – 42.5

Viable Hard 0.0 (0.0) 0.2 (0.1) 0.0 – 0.4

Dead 2.7 (0.8) * 1.3 (0.4) 0.3 – 2.4

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0 – 0.1

Note: The data were analyzed according to three ra ndomized complete block (RCB) designs with four replications; each site re presented a separate RCB. *Indicates a statistically signi ficant difference between MON 87705 and the control (p < 0.05). †No statistical comparison could be made due to lack of variability in the data.

1 For the alternating temperature regimes of 10/20, 10/30, or 20/30 °C, the lower temperature was maintained for 16 hours, and the hi gher temperature for eight hours.

2 Means based on four replicates (n = 4) of approximately 100 seeds. In some instances, the total percentage for both MON 87705 and the control did not equal exactly 100% due to numerical rounding of

the means. S.E. = Standard Error.

3 Minimum and maximum means determined from among the eight commercially available reference soybean varieties produced at the sites.Monsanto Company 09-SY-201U Page 122 of 471 VIII.D.2. Field Phenotypic, Agronomic Characteristics and Environmental

Interactions

Plant growth, development, and yield character istics were assessed und er field conditions as part of the plant characterization a ssessment of MON 87705. These data were developed to provide APHIS with a detailed characterization of MON 87705 relative to the conventional soybean c ontrol, A3525, and commerci ally available soybean. According to CFR § 340.6, as part of the pe tition to seek deregula tion, a petitioner must submit “A detailed description of the phenotype of the regulated article.” This information is being provided to assess whet her there are phenotypi c differences between MON 87705 compared to the unmo dified recipient organism that may impact its pest potential. Environmental intera ctions were also assessed as an indirect indicator of phenotypic changes to MON 87705 relative to the same comparators described above. The purpose of these field evaluations wa s to assess the phenotypic and agronomic characteristics and the plant-inse ct, plant-disease, or plant-abio tic stressor interactions of MON 87705 compared to the cont rol. Certain growth, reprod uction, and pre-harvest seed loss characteristics (such as lodging and pod sha ttering) can be used in the assessment of whether MON 87705 has enhanced plant pest potential.

Data were collected at 17 field locations during 2007 to t horoughly evaluate phenotypic, agronomic, and environmental interaction charac teristics. These 17 locations provided a diverse range of environmental and agronomic conditions representative of commercial soybean production areas in the U.S. (Table VIII-3). The experiments were arranged as randomized complete block designs. Th e categories and timi ngs of phenotypic

characteristics and environmental interactions evaluated are included in Table VIII-1.

The methods and detailed results of the indi vidual site data comparisons are presented and discussed in Appendix G, while the combined-site analyses are summarized below.

The results of this assessment showed th e improved fatty acid profile and glyphosate tolerance trait did not unexp ectedly alter MON 87705 compar ed to conventional soybean in terms of weediness potential, and the lack of differences in plant response to abiotic stressors, disease damage, arthropod damage, and arthropod pest an d beneficial insect abundance further support the c onclusion that the introduction of the modified oil profile trait is unlikely to increase plant pest potential.

VIII.D.2.1. Field Phenotypic and Agronomic Characteristics

A total of 14 phenotypic and agronomic char acteristics were evaluated. For the combined-site analyses, no significant di fferences were detected between MON 87705 and the control for seedling vigor, plant hei ght, lodging, pod shatte ring, seed moisture, test weight, or yield (Table VIII-4). F our statistically significant differences were

detected between MON 87705 and the contro l in the combined site analysis. MON 87705 was lower than the control for both early stand count (235.1 vs. 256.1 plants/plot) and final stand count (216.3 vs. 239.3 plants/plo t); however, mean counts of MON 87705 were within the reference soybean varieties range for both stand count evaluations. Furthermore, it is unlikely th at decreased stand count s would contribute to increased weed potential of MON 87705 and the detected differences in stand count did not impact final yield. MON 87705 flowered approximate ly one day later than theMonsanto Company 09-SY-201U Page 123 of 471 control (198.1 vs. 196.9 days after 1 Jan. 2007) and the weight of 100 seeds was lower for MON 87705 compared to the control (15.6 vs. 16.1 g). The differences in days to 50% flowering and 100 seed wei ght were relatively small in magnitude (0.6% and 3.2%, respectively), and the mean values of MON 877 05 were within the range of the reference soybean varieties for both characteristics. Thus , the differences in days to 50% flowering and 100 seed weight are unlikely to be biol ogically meaningful in terms of increased weed potential.

Flower color, plant pubescence, and plant grow th stage data were categorical and were not statistically analyzed; however, at each s ite all plants of MON 87705 and the control had purple flowers and pubescence as exp ected. Furthermore, MON 87705 and the control were within the same range of plan t growth stages for 113 out of the 114 growth stage observations among the si tes. During a single obser vation at one site, MON 87705 plants were at the V6 growth stage while the control plants were at V7; however, the growth stage of MON 87705 was within the ra nge of growth stages observed for the reference soybean varieties (Appe ndix G; Table G-4).

The phenotypic and agronomic characteristics evaluated in this study were used to provide a detailed descript ion of MON 87705 compared to the nontransformed control (A3525). A subset of these characteristics was useful to assess the w eediness potential of MON 87705 compared to the conventional soyb ean control. Based on the measured phenotypic and agronomic char acteristics, the re sults support a conclusion of no unexpected changes in the phenotype and no increased plant pest potential of MON 87705 compared to the conve ntional soybean control.Monsanto Company 09-SY-201U Page 124 of 471 Table VIII-3. Field Phenotypic Evaluation Sites for MON 87705 during 2007

Location † Location

Code USDA-APHIS

Notification Number Jackson County, Arkansas * AR 07-043-105n

07-115-103n Jefferson County, Iowa IA1 07-043-104n Benton County, Iowa IA2 07-043-104n Clinton County, Illinois IL1 07-043-104n Stark County, Illinois IL2 07-043-104n Warren County, Illinois IL3 07-043-105n Boone County, Indiana IN1 07-043-104n Parke County, Indiana IN2 07-043-105n Pawnee County, Kansas KS 07-043-105n Ottawa County, Michigan MI 07-043-105n Lincoln County, Missouri MO2 07-043-105n St. Louis County, Mi ssouri MO3 07-043-105n Macon County, Missouri MO4 07-043-105n York County, Nebraska NE 07-043-104n Fayette County, Ohio OH 07-043-104n Berks County, Pennsyl vania PA 07-043-104n Walworth County, Wisconsin WI 07-043-104n

* The Arkansas site utilized two USDA-APHIS notifications. 07-043-105n was a release

notification and 07-115-103n was a movement notification. †Shelby County, Missouri (USDA-APHIS notifi cation number 07-043-105n) data not reported due to wild animal damage early in the season.Monsanto Company 09-SY-201UPage 125 of 471Table VIII-4. Plant Growth and Development Data across 17 Locations during 2007MON 87705* A3525 Reference Range1Phenotypic Characteristic (units)Mean (S.E.) Mean (S.E.) Minimum MaximumEarly stand count (#/plot)235.1* (8.8) 256.1 (6.9) 142.3 267.2Seedling vigor (1-9 scale)3.0 (0.2) 2.9 (0.2) 2.3 5.4Days to 50% flowering2198.1* (1.1) 196.9 (1.1) 194.5 200.1Plant height (in)34.2 (0.9) 35.1 (0.9) 26.6 43.0Lodging (0-9 scale)1.8 (0.3) 1.6 (0.3) 0.4 2.3Pod shattering (0-9 scale)0.2 (0.1) 0.2 (0.1) 0.0 0.5Final stand count (#/plot)216.3* (8.7) 239.3 (7.3) 145.0 258.2Seed moisture (%)12.3 (0.3) 12.3 (0.3) 11.0 13.6100 seed weight (g)15.6* (0.4) 16.1 (0.4) 13.8 20.5Test weight (lb/bu)54.3 (0.6) 54.0 (0.7) 51.7 56.0Yield (bu/ac)54.3 (2.5) 54.8 (2.3) 38.7 63.8* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (A3525) (p < 0.05).1Reference range = Minimum and maximum mean values among the 17 commercially available reference soybean varieties.2Calendar day number when approximately 50% of the plants ineach plot were flowering.S.E. = standard error. Means based on n = 51 (except 100 seed weight where n = 48).Monsanto Company 09-SY-201U Page 126 of 471 VIII.D.2.2. Environmental Interaction Analyses

APHIS considers the environmental intera ction potential of the biotechnology-derived soybean compared to its convent ional counterpart to determin e the potential for increased weedy or invasive characteristics. Evalua tions of environmental interactions were conducted as part of the plant characteri zation for MON 87705. In the 2007 U.S. field trials conducted for evaluation of phenot ypic and agronomic characteristics of MON 87705, observational data on plant response to abioti c stressors (drought, wind, nutrient deficiency, etc.), disease dama ge, arthropod damage, and arthropod abundance (Appendix G; Tables G-5, G-6, G-7, G-8, and G-9, respectively) also were collected. These data are used as part of the environmen tal risk assessment to assess the plant pest potential and potential increased adverse im pact on NTOs for MON 87705 compared to the conventional soybean control (see Se ction X and Section XI for additional discussion). In addition, multiple commercia l soybean varieties were included in the analysis to establish a range of natural variability for each assessed characteristic. The

environmental interactions evaluation included data collected in the phenotypic studies (plant-insect, plant-disease, and plant-enviro nment interactions). The results of this assessment showed the improved fatty acid pr ofile and glyphosate to lerance trait did not unexpectedly alter MON 87705 compared to c onventional soybean in terms of pest potential. The lack of differences in plant re sponse to abiotic stressors, disease damage, arthropod damage, and arthropod pest and bene ficial insect abundanc e indicate that the introduction of the improved fa tty acid profile and glyphosate tolerance trait is unlikely to be biologically meaningful in terms of in creased pest potential. In these trials, the observations of plant response to abioti c stressors, disease damage, and arthropod damage were performed four times during the growing season at all 17 sites, and arthropod abundance was assessed from coll ections performed three times during the growing season at four of the 17 sites. The obs erved stressors were at natural levels (i.e.,

no artificial infestation or inte rference was used). Therefore, the same stressors were not necessarily observed at each field site.

Environmental interactions were assessed qualitatively at 17 s ites, and arthropod

abundance data were collected quantitatively from four s ites. For the plant-insect interactions, plant-disease interactions, a nd plant responses to abiotic stressors, the reported values represent the range of ratings observed across the three replications at

each site. MON 87705 and the control were considered qualitatively different in response to a stressor if th e ratings between MON 87705 a nd the conventional soybean control did not overlap across a ll replications for that part icular stressor (e.g., “none” rating vs. “slight-moderate” rating). Th e ratings observed among the commercial reference soybean varieties provide qualitati ve assessment data common to soybean for

each stressor assessed.

In an assessment of abiotic stress respons e, disease damage, and arthropod damage, no

differences were detected between MON 87705 and the conventional soybean control for 574 of 579 comparisons (including 167 abiotic stress response, 206 disease damage, and 206 arthropod damage comparisons) among all ob servations at the 17 sites (Appendix G; Tables G-5, G-6, a nd G-7). The five observed differe nces were in the disease and arthropod damage categories. MON 87705 ha d less damage than the control fromMonsanto Company 09-SY-201U Page 127 of 471 bacterial blight during three obs ervations at a single site a nd from aphids and leafhoppers during one observation at a single site. For each of the five observed differences, the severity of damage in the MON 87705 plots was within the range of th at for the reference soybean variety plots. Therefore, the de tected differences in disease and arthropod damage ratings are unlikely to be biologically meaningful in terms of increased plant pest potential for MON 87705 compared to th e conventional soybean control.

In an assessment of pest and beneficial arthropod abundance, no st atistical differences

were detected between MON 87705 and the c onventional soybean control for 95 out of 96 comparisons (including 46 arthropod pest comparisons and 50 be neficial arthropod comparisons) among the collection intervals at the four sites (Appendix G; Tables G-8 and G-9). The single detected statistically significant difference was for bean leaf beetle in a single collection from one site, and the mean abundance value from the MON 87705 plots was within the range of that for the reference soybean variety plots. Thus, the differences are unlikely to be biologically meaningful in terms of increased plant pest potential.

These results indicate that compared to conventional soybean, the environmental interactions between MON 87705 and arthropod pe st and beneficial or ganisms, diseases,

and abiotic stressors were not altered compared to conventional soybean. The lack of significant biological differences in plant re sponse to abiotic stressors, disease damage, arthropod damage, and arthropod pest and bene ficial insect abundanc e indicate that the improved fatty acid profile and glyphosate tole rance trait of MON 87705 is unlikely to be biologically meaningful in terms of increased plant pest potential.

VIII.D.3. Pollen Characteristics

APHIS considers the potential for gene flow to, and introgression of the biotechnologyderived trait into other soybean varieties and wild relatives to determine the potential for increased weedy or invasive characteristics of the receiving species. Pollen morphology and viability information are pertinent to th is assessment and therefore were assessed on MON 87705. In addition, morphological characterization of pollen produced by

MON 87705 and the conventional soybean control is relevant to the plant pest risk

assessment because it adds to the detailed description of the phenotype of MON 87705 compared to the conventional soybean.

The purpose of this evaluation was to as sess the morphology and viability of pollen collected from MON 87705 compared to a conve ntional soybean control. Pollen was collected from MON 87705, the control (A 3525), and five commercially available reference soybean varieties grown under simila r agronomic conditions in a field trial in Missouri. The trial was arranged in a ra ndomized complete bloc k design with three replications. A minimum of 20 flowers we re collected from each plot. Pollen was extracted, combined among flowers collected from the same plot, and stained with Alexander’s stain (Alexander, 1980). Pollen viability was evaluated for each sample and pollen grain diameter was measured for ten representative viable pollen grains per replication. General morphology of the pollen was observed for each of the three replications of MON 87705, the control, and the reference s oybean varieties (see Appendix H).Monsanto Company 09-SY-201U Page 128 of 471 No statistically significant differences were detected between MON 87705 and the control for percent viab le pollen or pollen grai n diameter (Table VIII-5). Furthermore, no visual differences in general pollen morphology were observed between MON 87705

and the control. These results demonstrat e that the introduction of the modified oil profile trait did not alter the overall morphology or viability of MON 87705 pollen compared to the conventional soybean control. The lack of statistically significant differences between the polle n collected from MON 87705 compared to the conventional soybean control for the assessed characteris tics demonstrate that the observed values were within the range of observations expected for soybe an. Thus, these data further support no change in plant pest pot ential for MON 87705 compared to the

nontransformed control and ot her soybean varieties.

Table VIII-5. Pollen Grain Diameter and Viability Analyses

Pollen

Characteristic MON 87705* A3525 Reference Range 1

Mean (S.E.) Mean (S.E.) Minimum Maximum

Viability (%) 98.3 (0.3) 96.7 (0.5) 97.2 99.6

Diameter (μm) 25.7 (0.1) 25.0 (0.5) 24.3 25.8

S.E. = Standard Error. Means based on n = 3. * No significant differences were detected between the MON 87705 and the control (p > 0.05).

1 Reference range is the minimum and maximum mean value observed among the five reference soybean

varieties.Monsanto Company 09-SY-201U Page 129 of 471 VIII.D.4. Symbiont Interactions

Members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae form a highly complex and specific symbiotic relationship with leguminous plants, including soybean (Gage, 2004). The nitrogen-fixing plant-micr obe symbiosis results in the formation of root nodules, providing an environment in whic h differentiated bacter ia called bacteroids are capable of reducing or fixi ng atmospheric nitrogen. The product of nitrogen fixation, ammonia, then can be utilized by the plant. In soybean, atmospheric nitrogen is fixed into ammonia through a symbiotic association with the bacterium Bradyrhizobium
japonicum . As a result of this relationship, no nitrogen inputs are needed for agricultural production of soybean.

As part of the plant pest risk assessment, APHIS considers the impact of the modified crop on pest potential and the environment as well as on agricultural or cultivation practices (CFR § 340.6). Changes in the symbiotic relationship with Rhizobiaceae and Bradyrhizobiaceae could directly impact pest potenti al, the environment, or cultivation practices (i.e., need to add additional nitrog en to soybean production). Thus, the purpose

of this evaluation was to assess whether the B. japonicum- soybean symbiosis of

MON 87705 had been altered as a result of th e introduction of the improved fatty acid profile and glyphosate tolerance trait compared to a convent ional soybean control.

The relative effectiveness of the symbiotic association be tween a leguminous plant and its rhizobial symbiont can be assessed by various methods. Assessment of nodule number and mass along with plant growth an d nitrogen status ar e commonly used to assess differences in the symbiotic associ ation between a legume and its associated rhizobia (Israel et al., 1986). It should be noted, however, that nodule number relative to nodule dry weight may be variable in soybean experiments because some nodules may be larger in diameter and less numerous, while others are not as developed (smaller) but more abundant (Appunu and Dhar, 2006; Israel et al., 1986).

MON 87705, a conventional soybean contro l (A3525), and six reference soybean varieties were produced from seed planted in pots containing nitrogen-deficient potting medium grown in a greenhouse. Seeds were inoculated with a solution of B. japonicum

. The pots were arranged in a ra ndomized complete block design with eight rep licates. At six weeks after emergence, plants were exci sed at the surface of the potting medium, and then shoot and root plus nodul e material were removed from the pots. Nodules were separated from roots prior to enumeration and determination of dry weight. Detailed

information on materials and methods used fo r symbiont evaluation is presented in Appendix I.

No significant differences were detected between MON 87705 and the control for each measured parameter, including nodule number, shoot total nitrogen (percent and mass), and biomass (dwt) of nodules, shoot material, and root material (Table VIII-6).

Based on the assessed characteristics, th e results support the conclusion that the introduction of the improved fatty acid prof ile and glyphosate tole rance trait does not alter the symbiotic relationship between B. japonicum and MON 87705 compared to conventional soybean. Thus, there is no incr eased plant pest poten tial and no expectedMonsanto Company 09-SY-201U Page 130 of 471 impact to cultivation practices relative to nitrogen inputs for MON 87705 compared to

the nontransformed control or other soybean varieties.

Table VIII-6. Symbiont Interaction Assessment of MON 87705 and the Control

Note: Pots were arranged in eight replicated blocks (n = 8) in a greenhouse using a randomized completed block design. S.E. = standard error. *No significant differences were detected between MON 87705 and the control (p>0.05).

1 Reference range is the minimum and maximum mean value observed among six commercial reference
soybean varieties.

VIII.E. Overall Conclusions for Phenotypic, Agronomic, and Environmental

Interactions Evaluation

An extensive and robust set of information and data were used to assess whether the introduction of the improved fatty acid profil e and glyphosate tolera nce tolerance trait altered the plant pest potential of MON 87705 compared to the conventional soybean control A3525. Phenotypic and agronomic ch aracteristics of MON 87705 were evaluated and compared to those of the conventional s oybean control. These assessments included 14 plant growth and development characteris tics; five seed dormancy and germination parameters under six different temperature regimes; tw o pollen characteristics; observations for abiotic stressor, diseas e damage, arthropod damage and arthropod abundance; plant-symbiont interaction char acteristics; and com positional evaluation (Section VII) of 67 different components (s even in forage, and 60 in seed).

Results from the phenotypic and agronomic assessments demonstrate that MON 87705 does not possess characteristics that would c onfer a plant pest risk compared to conventional soybean. Data on environmental interactions also i ndicate that MON 87705 does not confer any biologically meaningful increased suscep tibility or tolerance to specific disease, insect, or abiotic stressors, or change s in agronomic and phenotypic characteristics. Taken together, these data support the conclusion that MON 87705 is not likely to pose increased plant pest ri sk compared to conventional soybean. Measurement
Endpoint Mean (S.E.)* Reference Range 1

MON 87705 A3525 p-Value Min Max

Nodule Number
(per plant) 229 (43) 246 (39) 0.6625 140 235

Nodule Dry Wt (g/plant) 0.61 (0.08) 0.64 (0.03) 0.7160 0.52 0.73

Root Dry Wt (g/plant) 1.68 (0.11) 1.52 (0.10) 0.4228 1.54 2.22

Shoot Dry Wt (g/plant) 5.24 (0.59) 5.13 (0.38) 0.8839 5.76 7.71

Shoot Total Nitrogen
(% dwt) 3.26 (0.21) 3.54 (0.12) 0.0832 2.74 3.41

Shoot Total Nitrogen (g) 0.18 (0.02) 0.18 (0.02) 0.7957 0.18 0.23Monsanto Company 09-SY-201U Page 131 of 471 IX. U.S. AGRONOMIC PRACTICES

IX.A. Introduction

As part of the plant pest assessment required by 7 CFR § 340.6(c)(4) impacts to agricultural and cultivation practices must be considered. This section provides a summary of current agronomic practices in the U.S. fo r producing soybean and is included in this petition as a baseline to a ssess possible impacts to agricultural practices

due to the cultivation of MON 87705. Di scussions include soybean production, seed production, growth and devel opment, general management practices (including identity preservation practices), management of weed s, insects and diseases, soybean rotational crops, and volunteer soybean management. Info rmation presented in the previous section

demonstrated that MON 87705 is no more su sceptible to diseas es or pests than conventional soybean. Additionally data pres ented in Section VIII show that, with the exception of an improved fatty acid profile and tolerance to the herbicide glyphosate, MON 87705 is phenotypically e quivalent to conventional s oybean. Thus, there are no changes to the inputs needed for MON 87705, and no specific impacts to most of the agronomic practices employed for production of soybean. Wh ere there is potential for impact on agronomic practices from the deregulation of MON 87705, discussion delineating the scope and magn itude of those impacts is provided. For example, MON 87705 will be produced unde r an identity pres ervation system re quiring specific management practices to preserve the value of the oil. Th erefore, emphasis is placed on anticipated impacts to agronom ic practices used for producti on of specialty type soybean uponderegulation of MON 87705. Additionally MON 87705 has a glyphosate-tolerance trait so potential impacts to crop rotation practices are considered.

Soybean is planted in over 30 states, demons trating its wide adaptation to soils and climate. The soil, moisture, and temper ature requirements for producing soybean are generally similar to those for corn, and thus th e two crops share a sim ilar cultivation area. Proper seedbed preparation, appropriate variet y selection, appropria te planting dates and plant population, and good integrated pest management practices are important for optimizing the yield potential and economic returns of soybean.

MON 87705 is expected to bring added value for growers, soybean handlers, crushersand food processors. An identity preservation system will be used for production, postharvest handling and processing to preserve the enhanced value of MON 87705. As such, production practices, post-harvest handling of soybean, and processing will fall under a separate production system a nd distribution channel termed for this specialty soybean. MON 87705 is expected to utilize existing ag ricultural practices employed for production and identity preservation of specialty soybean.

Annual and perennial weeds are perceived to be the greatest pest problem in soybean production. Economic thresholds for controllin g weeds in soybean require some form of weed management practice on all soybean acre age. Approximately 98% of the soybean acreage receives a herbicide application. Soybean insects and diseases generally areMonsanto Company 09-SY-201U Page 132 of 471 considered less problematic, although infest ations can reach economic thresholds requiring treatment.

Volunteer soybean, i.e., soybean plants that have germinated and emerged unintentionally in a subsequent crop, are not considered a significant concern in rotational crops primarily because of climatic conditions an d adequate control from tillage practices. Additionally, mechanical and chemical control methods ar e available to manage the occasional volunteer soybean plant. Due to the lack of weediness potential, introduction of MON 87705 in the soybean production system would have a negligible impact on managing soybean volunteer plants in rotation al crops such as corn, cotton, and rice,

because control measures are available for volu nteer soybean when they arise. Preplant tillage is the first management tool for control of emerging volunteer soybean in the spring. If volunteer soybean pl ants emerge after planting, sh allow cultivation will control most of the plants and effectively reduce competition with the crop. Several postemergence herbicides also are available to control volunteer soybean (conventional or glyphosate-tolerant soyb ean) in each of the ma jor rotational crops.

As shown in Sections VII and VIII, with th e exception of the improved fatty acid profile and glyphosate tolerance trait, no phenotypic, compositional, or environmental

differences between MON 87705 and conventional soybean have been observed. Moreover herbicide-tolerant soybean is currently grown on 92% of soybean acres (USDA-NASS, 2008). Therefore, it is not anticipated that commercialization of

MON 87705 in the U.S. would have a nota ble impact on current soybean cultivation practices, including the management of weeds, diseases, and insects.

IX.B. Overview of U.S. Soybean Production

IX.B.1. Soybean Production

Soybean first entered North America in the 18 th century (Hoeft et al., 2000). Sometime during the 1930s, soybean started to be proce ssed industrially in the U.S. for edible oil and protein meal. Currently, the U.S. produ ces approximately 32% of the global soybean supply (ASA, 2008). In 2007, the U.S. exporte d 1 billion bushels (27.9 million metric tons) of soybean, which accounted for 37 perc ent of the world's soybean exports (ASA, 2008). In total, the U.S. exported $12.9 billion USD worth of soybean and soybean products globally in 2007 (ASA , 2008). China is the larges t export market for U.S. soybean with purchases totalling $4.1 billion. Japan is the second largest export market with sales of $1.1 billion in the same year . Other significant markets include the European Union and Mexico.

The production of soybean is hi ghly dependent upon soil and climatic conditions. In the U.S., the soil and climatic requirements for gr owing soybean are very similar to corn. The soils and climate in the Eastern, Midweste rn and portions of the Great Plains regions of the U.S. provide sufficient water supplie s under normal climatic conditions to produce a soybean crop. The general water requireme nt for a high-yielding soybean crop is approximately 20 inches of water during the growing season (Hoeft et al., 2000). Soil texture and structure are key components dete rmining water availability in soils, where

medium-textured soils hold more available water, allowing soybean roots to penetrateMonsanto Company 09-SY-201U Page 133 of 471 deeper in medium-textured soils than in clay soils. Irrigation is used on approximately 9% of the acreage to supplement the water su pply during dry periods in the western and southern soybean growin g regions (ASA, 2008).

Most of the soybean acreage is grown as a full-season crop. Approximately 8% of the soybean acres are planted in a double-crop sy stem following winter wheat south of 35o North latitude (Boerma and Specht, 2004)). However, this percentage can vary significantly from year to y ear. The decision to plant doubl e-crop soybean is influenced by both agronomic and economic fa ctors. Agronomic factors in clude harvest date of the wheat crop, which determines the double-crop soybean planting date , and available soil

moisture. Economic factors include expected soybean price and anticipated economic return (Boerma and Specht, 2004).

The vast majority of soybean grown in the U. S. is grown for animal feed and is usually fed as soybean meal. However, soybean is also grown as a specia lty soybean product for a specific market or use. 5 Examples of specialty soybean include high protein, tofu, high oil, high oleic, non-biotechnology- derived (also referred to as non-genetically modified), and organic soybean. 6 The uses of these soybean varieties include human consumption, food processing or specialty products. The soyb ean varieties used in the specialty market are typically specified by buyers and end-us ers of soybean for pr oduction, and a premium relative to commodity soybean is paid fo r delivering a product that meets purity and quality standards (Pri tchett et al., 2002; Elberhi, 2007; Sundstrom et al., 2002; Lee and Herbek, 2004; Muth et al, 2003; and Smyth and Phillips, 2002) for th e soybean variety. Product differentiation and market segmenta tion in the specialty soybean industry includes mechanisms to keep track of the soybean (traceab ility), methods for identity preservation (IDP), including closed-loop system s, and quality assurance processes (e.g., ISO9001-2000 certification), as well as cont racts between grower s and buyers that specify delivery agreements. MON 87705 is an improved fatty acid profile soybean oil product and will be considered a specialty soybean product and marketed in a manner similar to other high-value specialty crops.

The U.S. soybean acreage in the past 10 year s has varied from appr oximately 64.7 to 75.7 million acres, with the lowest acreage recorded in 2007 and the highest in 2008 (Table IX-1). Average soybean yields have varied from 33.9 to 43.3 bushels per acre over this same time period. Soybean production ranged fr om 2.45 to 3.19 billi on bushels over the past ten years, with 2006 bei ng the largest production year on record. According to data from USDA-NASS (2009a,b), soybean was pl anted on approximately 75.7 million acres in the U.S. in 2008, producing 2.96 billion bushe ls of soybean (Table IX-1). Soybean acreage and production in 2007 wa s down significantly from 2006, mainly due to a large increase in corn acreage. The value of soyb ean reached $27.4 billion in the U.S. in 2008 (USDA-NASS, 2009a,b). In comparison, corn and wheat values in 2008 were $47.37 and $16.57 billion, respectiv ely (USDA-NASS, 2009a,b).

5 http://usb.adayana.com:8080/usb/jsp/login.jsp

6 http://usb.adayana.com:8080/usb/jsp/login.jspMonsanto Company 09-SY-201U Page 134 of 471 For purposes of this agronomic practices di scussion, soybean production is divided into three major soybean growing regions accounting for 99.1% of the 2008 U.S. soybean acreage: Midwest/Great Plains region (IL , IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, SD, and WI), Southeast regi on (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern Coastal region (DE, MD, NJ, NY, PA, and VA) (Table IX-2). The vast majority of soybean was grown in the Midw est region, representing 82.1% of the total U.S. acreage. The Southeast and Eastern Coastal regions re presented 14.3% and 2.7% of the acreage, respectively. Among the three regions, the Midwest region produced the highest average yield at 38.6 bushels per acre in 2008, and aver age state yields in this region ranged from 28.0 to 47.0 bushels per acre. The average yield in the S outheast region was 34.4 bushels per acre, with states within this region aver aging from 30.0 to 40.0 bushels per acre. The average yield in the Eastern Coastal region was 34.1 bushels per ac re, with individual state averages ranging from 27.5 to 46.0 bushels per acre.

Managing input costs is a ma jor component to the economi cs of producing a soybean crop. Key decisions on input costs include choosing what soybean varieties to plant, amounts of fertilizer to apply, and what herb icide program to use. The average operating cost for producing soybean in the U.S. in 2006 was $93.41 per acre, according to statistics compiled by the USDA-Economic Re search Service (USDA-ERS, 2006). The value of the produc tion less operating cost was report ed to be $161.43 per acre. A summary of potential produc tion costs and returns are pr esented in Table IX-3.

Table IX-1. Soybean Production in the U.S., 1999 – 2008

Year Acres

Planted (×1000) Acres

Harvested

(×1000) Average

Yield (bushels/acre) Total

Production (×1000 bushels) Value (billions $)

2008

2007 75,718 64,741 74,641 64,146 39.6

41.7 2,959,174 2,677,117 27.40

26.97 2006 75,522 74,602 42.7 3,188,247 20.42 2005 72,142 71,361 43.3 3,086,432 16.93 2004 75,208 73,958 42.2 3,123,686 17.89 2003 73,404 72,476 33.9 2,453,665 18.01 2002 73,963 72,497 38.0 2,756,147 15.25 2001 74,075 72,975 39.6 2,890,682 12.61 2000 74,266 72,408 38.1 2,757,810 12.47 1999 73,730 72,446 36.6 2,653,758 12.21
Source: USDA-NASS, 2009b.Monsanto Company 09-SY-201U Page 135 of 471 Table IX-2. U.S. Soybean Production by Region and State in 2008

Region/State Acres

Planted 1
(thousands) Acres

Harvested 1
(thousands) Average Yield 1
(bushels/acre) Total

Production 1
(×1000 bushels) Value 1
(billions $)
Midwest Region

Illinois 9,200 9,100 47.0 427,700 4.00

Indiana 5,450 5,430 45.0 244,350 2.27

Iowa 9,750 9,670 46.0 444,820 4.29

Kansas 3,300 3,250 37.0 120,250 1.03
Kentucky 1,390 1,380 34.0 46,920 0.42
Michigan 1,900 1,890 37.0 69,930 0.64

Minnesota 7,050 6,950 38.0 264,100 2.54

Missouri 5,200 5,030 38.0 191,140 1.72

Nebraska 4,900 4,860 46.5 225,990 2.12

North Dakota 3,800 3,760 28.0 105,280 0.96

Ohio 4,500 4,480 36.0 161,280 1.55

South Dakota 4,100 4,060 34.0 138,040 1.25

Wisconsin 1,610 1,590 35.0 55,650 0.51

Region Totals 62,150 61,450 38.6 2,495,450 23.30
Southeast Region

Alabama 360 350 35.0 12,250 0.12

Arkansas 3,300 3,250 38.0 123,500 1.09

Georgia 430 415 30.0 12,450 0.11

Louisiana 1,050 950 33.0 31,350 0.29

Mississippi 2,000 1,960 40.0 78,400 0.69

North Carolina 1,690 1,670 33.0 55,110 0.47

South Carolina 540 530 32.0 16,960 0.15

Tennessee 1,490 1,460 34.0 49,640 0.43

Region Totals 10,860 10,585 34.4 379,660 3.35
Eastern Coastal Region

Delaware 195 193 27.5 5,308 0.05

Maryland 495 485 30.0 14,550 0.13

New Jersey 92 90 29.0 2,610 0.02

New York 230 226 46.0 10,396 0.09
Pennsylvania 435 430 40.0 17,200 0.15
Virginia 580 570 32.0 18,240 0.16

Region Totals 2027 1994 34.1 68,304 0.60

1 Source: USDA-NASS, 2009b.Monsanto Company 09-SY-201U Page 136 of 471 Table IX-3. U.S. Soybean Production Costs and Returns in 2006

Production Cost or Return Cate g or y Itemized Costs Return per
Planted Acre ($ USD)

Total Gross Value of Production 254.84

O p eratin g Costs: Seed 32.30

Fertilize r 13.05

Chemicals 14.46

Custom o p erations 6.01
Fuel, lube and electricit y 13.51

Re p airs 11.80

Purchased irri g ation wate r 0.11

Interest on o p eratin g ca p ital 2.17
Total, o p eratin g costs 93.41

Allocated overhead: Hired labo r 1.78 Opportunity cost of unpaid

g rower’s labo r 15.20

Capital recovery of machinery
and e q ui p men t 60.38

Opportunity cost of land (rental rate) 86.17

Taxes and insurance 7.93

General farm overhead 13.22
Total, allocated overhead 184.68

Total cost listed 278.09

Value of production less total cost
listed (23.25)

Value of production less operating

costs 161.43

Supporting Information: Yield = 46 bushels/acre, Price = $5.54/bushel, Enterprise size = 268 planted acres, Irrigated = 9%, Dry land = 91%.

Source: USDA-ERS, 2006.Monsanto Company 09-SY-201U Page 137 of 471 IX.B.2. Specialty Soybean

Commodity and specialty s oybean are the two primary production and distribution systems for soybean produced in the U.S. The majority of soybean is commonly grown and marketed through commodity markets for the oil and protein content. Commodity soybean is not consumed directly, but is crus hed for meal that is predominantly used for animal feed and as a minor protein source fo r food. The oil produced during the crushing of the soybean is used for cooking or food ingredients. The goa l of the commodity supply chain is to supply a homogenous produc t to the enduser. The grower producing soybean for this chain has a choice from many different vari eties for production; however, harvested soybean is viewed to be the same for all commodity soybean varieties. At harvest, the gr ower either delivers soybean to a handler or stores them on farm for later delivery. The handler is not interested in differentiating the commodity soybean for later use. Commodity soybean ha ndlers typically have large volume storage capacity. Similarly, commodity soybean proc essors crush large vo lumes of soybean to produce homogeneous oil and meal products. The commodity system is designed to maximize efficiency at a low profit margin re sulting in comingling of different sources of soybean that does not affect the price receiv ed for the final product. This production system has been in pl ace in the U.S. since the production of soybean began in earnest in the 1960s (Sonka et al., 2004).

In recent years, there has been an increased demand by c onsumers and food processers for soybean that has specific physical or ch emical characteristics that are required by

certain customers to meet specific food or feed needs. As a result a separate specialty soybean channel has developed. This produ ction system and dist ribution channel is

focused on value-added traits that involve much smaller volumes than commodity soybean (Sonka et al., 2004). Specialty soyb ean varieties are produced on approximately 12% of the U.S. soybean acreage 7 (and according to the Midw est Shippers Association (MSA, 2009), this acreage could grow to over 25% of the crop acreage in certain states within the next decade. This supply chain typically consists of a specialty firm that contracts production of a specifi c variety and sets standards for quality of the harvested soybean (Lee and Herbek, 2004). In return , growers receive a premium over the price paid for commodity soybean. Growers may st ore harvested soybean on farm or deliver the product directly to a proce ssor or to special c ontainers for international shipment. The goal of this identity preserved system is to minimize handling so that value is maintained. The cost incurred from an identify preservation system is offset by the higher value received for the final product.

According to the American Soybean Asso ciation (2009), specialty soybean can be grouped into ten broad categories: non-biot echnology-derived, certified seed, organic food-grade, low saturated fat, clear hilum, tofu, natto, high sucrose, high oleic, low

7 http://usb.adayana.com: 8080/usb/jsp/login.jsp : Percent U.S. soybean acreage estimate based on

U.S. Domestic Consumption by Segment – 200 8/09. D. Ludwig, personal communication, 2009.Monsanto Company 09-SY-201U Page 138 of 471 linolenic and high protein. Th e categories refer to soybean w ith characteristics such as altered seed composition (e.g., low saturated fa t, high sucrose, high oleic, low linolenic, and high protein), varieties of soybean with unique physical characteri stics suited to their specific uses (e,g., clear hilum for direct human consumption), or refer to a production process (e.g., organic, certifie d seed). The categories are not meant to be exclusive; for example, soybean used to produce natto or tofu may employ organic production processes, and soybeans from all of the categories are of ten derived from varieties produced according to certified seed produc tion practices. Tofu and soymilk produced from the tofu soybean category represent a la rge segment of the speci alty soybean market and are produced from unique soybean varietie s that have clear hilum and large seed size (Lee and Herbek, 2004). Tofu varieties also mu st be high in protein (40% or higher) and low in oil concentration compared to co mmodity soybean. Clear hilum and other characteristic are required for soybean used in the production of other soybean food products consumed directly by humans su ch as natto, soybean sprouts, edamame (vegetable soybean), and soy nuts (Lee a nd Herbek, 2004; UK, 2009). Organic foodgrade and non biotechnology-deri ved soybean varieties are identity protected and produced not to contain biotec hnology-derived traits. Orga nic soybean have additional production restrictions requiring the soybean to be produced using no synthetic fertilizers or pesticides. In recent years, public and private soybean breeders have developed soybean varieties with improved nutritional ch aracteristics (e.g., high sucrose, high oleic, low linolenic and high protein) and MON 87705 is considered part of this trend towards production of value-added soybean products. The characteristics or modifications in this group include reducing the need for hydrogenati on of the soybean oil, increasing sugar concentration, increasing protein concentrati on, decreasing concentra tion of saturated and polyunsaturated fats, and lipoxygenase free soyb ean which removes some of the flavors that are objectionable to some c onsumers (Lee and Herbek, 2004).

The majority of specialty s oybean varieties are offered in Maturity Group II and early Group III varieties which are ad apted to the upper Midwest/Gr eat Plains region (Section IX.C) (Lee and Herbek, 2004). Ma turity Group II and III soybean varieties are grown on approximately 42-45 million acres, occupying the largest percentage of soybean acreage (see Section IX.C). The varieties were developed for this area primarily due to proximity to processing facilities as well as international routes of shipment and to take advantage of efficiencies in soybean breeding program s (S. Joehl, personal communication, April 2009). With a few exceptions, the agronomic or management practices for growing specialty soybean from planting to harvest are similar to commodity soybean (Lee and Herbek, 2004). Because special varieties ar e used in the production of each specialty soybean, the variety selection is dictated by the specialty s oybean buyer or processor.

Non biotechnology-derived and or ganic soybean by definition mu st be produced utilizing only conventional soybean varie ties. Weed control is extr emely important for specialty soybean to maintain a high yield potential and because weeds, such as nightshade ( Solanum nigrum ), can stain harvested soybean, which is particularly und esirable in foodgrade soybean (TCM, 2008). Be cause organic soybean must be grown without synthetic fertilizers or pesticides for three or more years prior to the current crop of soybean, fertilization and pest management is much more difficult (Lee and Herbek, 2004). Weed control in organic soybean relies on a comb ination of crop rotation, tillage, in-cropMonsanto Company 09-SY-201U Page 139 of 471 cultivation, and hand-weeding. Insect and disease control is managed primarily through crop rotation. Certain approved pesticides are permitted in this specialty soybean production.

All equipment and storage faci lities for specialty soybean mu st be clean of seed from other soybean varieties or pl ants, dirt, pathogens and othe r foreign material. Some soybean contracts may require a special inspec tion of the handling and storage facilities. The specialty soybean for soybean foods may require special harves ting equipment since some of these soybeans are harvested before full maturity (e.g., edemame or vegetable soybean).

IX.B.3. Identity Preservation

Identity Preservation (IDP) refers to a sy stem of production, handling, and marketing practices that maintains the integrity and purity of agricultural commodities (Sundstrom et al., 2002). Commodity grains, on the ot her hand, are marketed in mass according to USDA grading standards. Specialty crops requ ire some form of segregation or full-scale identity preservation to keep these grains separate from commod ity grains (Elberhi, 2007). This market segmentati on within the grain channeling system is driven by the need to preserve market value or ensure a specified purity of the product. With certain specialty crops, IDP is require d to prevent accidental or unintended commingling (e.g., non biotechnology, organic) or to segregate products that are approved only for certain uses (industrial use only).

IDP grain production has been in existence fo r a long time. Agricultural producers have over many decades developed practices that allowed for differentiation between food vs. feed grain, or grain vs. seed production, or organic vs. non organic (Massey, 2002). Seed certification programs such as those used by th e Association of Official Seed Certifying Agencies are often cited as th e model of IDP systems. These programs date back to the 1920s and 1930s when the certification process was implemented to verify the genetic purity of seed made availa ble to growers (see Section IX.B.4). Standards were established to ensure production of seed from known pedigree with high purity and quality. Similarly, commodity grain trad ers, marketing orga nizations, and food processors have established purity and quality tolerances for specific end-product uses. The need for segregation and IDP produc tion systems has in creased with the development of specialty crops or crops with special output traits, su ch as high oil corn, high oleic sunflower, and low-linolenic soybean (Sundstrom et al., 2002).

The production of IDP grains requires special processes in order for growers to meet buyers’ criteria for variety identification, composition, and quality. Buyers of IDP soybean typically contract for the production of seed-variety speci fic soybean and work directly with seed suppliers, growers, independent certification agencies, intermediate processors, and freight comp anies to deliver the prefer red product with in specified

tolerances. 8 Contract specifications are written to ensure the delivery of the desired

8 United Soybean Board, International Buyer’s Guide at http://www.ussoyexports.org/ussoy/buyersguide/Chap6.pdfMonsanto Company 09-SY-201U Page 140 of 471 attribute-specific product and that pred etermined management practices are used (Massey, 2002). Lack of compliance with a product specification can lead either to a price discount or rejecti on of a shipment by the buyer. Depending on the end use, contracts can be extensive, defining ma ny of the production, harvest and storage activities, or less stringent dealing more with pricing, quality specifications and only the most critical production practices . Premiums are paid to gr owers for the additional risk and management only when the grain meets the contract specificati ons. Premium prices provide the impetus to maintain the specialty grain’s purity and iden tity separate from commodity grains (Massey, 2002). Premiums are affected by various factors, including the proximity of suppliers to buyers and the cost and av ailability of substitutes (Sundstrom et al., 2002). For many trait-spec ific crops, price premiums rise or fall depending on the supply conditions for the generic commodity.

Growers of IDP grains must have good managerial ability and implement certain management practices to produce and deliver grain possessing the desired physical and chemical characteristics. The production and marketing of trait-spec ific grains involves additional financial risks (Elb erhi, 2007). The growers’ mana gerial ability can affect both yield performance and proficiency in m eeting the contract specifications. From a soybean buyer’s perspective, contracts he lp the buyer meet the demand for specific product qualities, improve cost efficiencies of product processing, and reduce transaction

costs.

While specific IDP production practices vary depending on the characteristics of the product to be delivered, general elements are im plemented to ensure that the end-user or processor receives the grain or end-product with the intended identity and desired quality. As mentioned above, many IDP systems were de veloped using the principles similar to those used in seed certification. IDP produc tion begins with a system of standards, records, and auditing that are put in place throughout the enti re crop production, harvesting, handling, and marketing pro cess (Sundstrom et al., 2002). Some key

considerations in the establishment of an IDP system include: 1) plan ted seed identity and tolerances, 2) appropriate fiel d isolation, 3) inspection an d clean-out of equipment and facilities, 4) end-product sampling and testing, and 5) record maintenance and identity labeling (Sundstrom et al., 2002). Each of these components are described in greater

detailed below:

Planting Seed Identity and Tolerances

The purity and identity of starting seed should be tested and confirmed. The purity of the seed stock should equal or exceed the purity standards of the desired final product. Single or multiple quality tolerances may be established in specialized IDP programs

based on market-driven standards.

Field Isolation

Crops must be isolated either spatially or temporally fr om pollen sources that could impact the quality or purity of the harveste d seed. The amount of isolation depends on flower characteristics, sexua l compatibility with neighboring crops, pollen quantity and viability, and mode of pollen dissemination. A self-pollinating crop, such as soybean, requires relatively small isolation distances to effectively preclude cross-pollination.Monsanto Company 09-SY-201U Page 141 of 471 Equipment and Facilities

Equipment used in production should be clea ned and inspected before and after use for IDP crop. Storage facilities and transporting ve hicles are cleaned and inspected to assure that segregation is maintained a nd no physical contamination occurs.

Sampling and Testing

In some cases, the IDP grain is sampled and tested at various stages to confirm the product identification, purity, and quality. Sp ecial consideration should be given to sampling and testing techniques th at ensure reliable results.

Record Maintenance and Identity Labeling

Records typically are maintained on field designations, harvest am ounts, storage bin locations, and product transfers. IDP products must be identified, segregated, and labeled

in the market chain.

Because value capture is also a vital part of IDP productions systems, growers must assure markets or buyers for these IDP crops ar e available, especially if the crop is not being grown under contract. The soybean industry has coll aborated to foster the development and availability of several specialty soybean and soybean oil markets. 9 As an example, the soybean industry initiated a program called the Low-Linolenic Locater, an internet-based program to assist growers in locating the closest processor or elevator that contracts acreage and offers pr emiums for low-linolenic soybean.

As with current IDP systems used for specialty soybean to preserve market value or ensure purity of the product, MON 87705 will employ an IDP system based on established practices. The IDP practices will be implemented to ensure that the end-user or processor receives the soybean with the id entity, fatty acid composition of the oil, and desired quality for this value-added specialty soybean.

IX.B.4. Soybean Seed Production

Standardized seed production practices are responsible for maintaining high-quality seed stocks, an essential basis for U.S. agricultu re. By the early 20th century, agronomists learned how to develop specific plant varieties with desirable traits. In the U.S., state agricultural experiment stations developed many seed varietie s that were distributed to growers for use. Seed was saved by grower s and later sold to ne ighbors; however, the

desirable traits of the varieties often we re lost through random genetic changes and contamination with other crop a nd weed seed (Sundstrom et al., 2002). The value of seed quality (including genetic purity, vigor, and presence of weed seed, seed-borne diseases, and inert materials, such as dirt) was quickly identified as a major factor in crop yields. States developed seed laws and certificati on agencies to ensure that purchasers who

received certified seed coul d be assured that the seed met established seed quality standards (Bradford, 2006). The federal govern ment passed the U.S. Federal Seed Act of 1939 to recognize seed cer tification and official certifying agencies. Regulations first

9 Qualisoy: Low-linoleic locator at http://www.qualisoy.com\Monsanto Company 09-SY-201U Page 142 of 471 adopted in 1969 under the Federal Seed Act recognize land history, field isolation, and varietal purity standards for foundation, registered, and certified seed. Under international agreements such as the Or ganization for Economic Co-Operation and Development (OECD) scheme, the U.S. an d other countries mutually recognize minimum seed quality standards (Bradford, 2006). The Association of Official Seed Certifying Agencies represents state and private seed cer tification in the U.S., and

includes international member countries in North and South America, Australia, and New

Zealand.

Soybean seed is separated into four seed cla sses: 1) breeder, 2) f oundation, 3) registered, and 4) certified (AOSCA, 2009a). Breeder seed is seed di rectly controlled by the originating or sponsoring plan t breeding organization or firm . Foundation seed is firstgeneration seed increased from breeder seed an d is handled to maintain specific varietal purity and identity. Registered seed is the pr ogeny of foundation seed that is handled to maintain satisfactory variety purity and identity . Certified seed is the progeny of breeder, foundation or registered seed, and is two ge nerations from foundati on seed. All soybean seed sold may not be officially certified; however, commercial soybean seed sold and planted for normal soybean production is prod uced predominately to meet or exceed

certified seed standards. This section of th e petition will provide a broad overview of the practices used in produc ing certified seed.

Soybean seed breeders and producers have put in place practical measures to assure the quality and genetic purity of soybean seed va rieties for commercial planting. The need for such systems arose from the recogniti on that the quality of improved soybean varieties quickly deteriorated in the absence of monitoring fo r quality and genetic purity (CAST, 2007). Seed certification programs we re initiated in the early 1900s in the U.S. to preserve the genetic identity and variet y purity of seed. Th ere are special land requirements, seed stock eligibility requirements, field inspections and seed labeling

standards for seed certificati on. Seed certification services are available through various state agencies affiliated with the Association of Official Seed Certifying Agencies. Large seed producers implement their own seed quali ty assurance programs. However, large seed producers often will utilize the services of state certifying agencies as a third party source to perform certain fiel d inspections and audits.

The U.S. soybean production for all purposes has varied from appr oximately 64.7 to 75.7 million acres in the past ten years, with the lowest acreage recorded in 2007 and the highest in 2008 (USDA-NASS, 2009 b; Table IX-1). This range of soybean acreage would require between 105 - 125 m illion units (50 lbs/unit) of soybean seed. This seed

volume includes allowances for seed losses due to weather, poor yields, and quality

issues. Additional allowances are included for distribution excess, seed returns, replants, and potential increases in soybean acreage. Assuming an average soybean yield of 45 bushels, or 54 units (50 lbs/unit) per acre, 1. 9 to 2.3 million acres would be required to produce this volume of commercial cert ified soybean seed each year.

Soybean seed is produced throughout most of the U.S. soybean-growing regions. Soybean varieties are developed and adapte d to certain geographical zones and are separated into ten maturity groups – Group 00 to Group VIII (see Section IX.C.). Seed production for these maturity groups is grow n in the respective geographical zone forMonsanto Company 09-SY-201U Page 143 of 471 each maturity group. However, the producti on areas generally are on the northern edge of the respective zone to minimi ze incidences of disease.

Soybean seed is produced by companies that produce and sell seed, such as Monsanto Company, Pioneer Hi-Bred International, Syngenta Seeds, Kruger Seed Co., Becks Hybrids, and tollers, which are companies that produce but do not sell certified seed, such as Remington Seeds LLC and Precision Soya. Seed companies and tollers in turn contract acreage with grow ers to produce the required amount of soybean seed. Production or processing plants at these seed companies id entify top soybean growers to produce the seed and also mon itor and inspect seed fields throughout the growing season. The production plants also clean, condition, an d bag the harvested soybean seed as well as monitor and inspect all the processes at the plant. Production plants typically produce between 100,000 units to 2,000, 000 units of soybean seed . Production plants will produce the various soybean varieties in differe nt climates or environments to spread production risks.

The entire seed production process at the majo rity of the seed comp anies and tollers is International Organization for Standardization (ISO) certifie d and; therefore, includes

internal and external audits (ISO, 2009). ISO standards ensure desirable characteristics of seeds and services, such as quality, safety, reliability, and efficiency. The ISO standards represent an international cons ensus on good management practices with the aim of ensuring that the organization can consistently deliver excellent product or services. The standards not only must meet the customer’s require ments and applicable seed regulatory requirements, but must aim to enhance customer satisfaction and achieve continual improvement of its performa nce in pursuit of these objectives.

The field operations and management practices for producing soybean seed are similar to normal soybean production. However, special attention is needed in certain areas to produce seed with high quality, high germination rates, and high genetic purity (Helsel and Minor, 1993). General guide lines specific for seed prod uction are discussed below. The seed production field should not have been planted with soybean the previous crop in order to avoid volunteer soybean plants (eve n though the risk of s oybean volunteer plants is negligible) and to ensure genetic purity.

Very early planting should be avoided becau se the seed produced from early planting often results in poorer quality seed (Helsel a nd Minor, 1993). Every effort must be made to eliminate weeds in a seed field through the use of herbicides and cultivation to prevent weed seed in the harvested soybean seed. Fields are scouted frequently for insect pests and insecticides are applied when insect pe st infestations reach economical threshold levels. Foliar-applied fungicides should be considered when disease infestations are predicted in the area. Harves t should occur as soon as the mature soybean seed reaches 13% moisture content. Harvesting soybean s eed with less than 13% moisture can cause damage to the seed coat and result in split soybean seed that can affect germination and viability. Harvesting equipment must be adjusted to minimize or avoid seed damage. Harvesting equipment must be cleaned befo re entering the seed fields to minimize genetic contamination. Certai n handling equipment, such as auger elevators, should be avoided because they can increase seed damage.Monsanto Company 09-SY-201U Page 144 of 471 Field inspections are vital to ensure th e soybean seed meets seed certification requirements, ISO certification standards, regulatory standards, and trait licensing agreement standards. Field inspections are conducted on seed production fields throughout the soybean growing season to evalua te variety purity, ensure soybean plants are developing properly, and fiel ds are maintained free of weed s, insects, and diseases. The fields are also mapped to ensure the seed field has the minimum federal isolation requirement of five feet (AOS CA, 2009a). Some states and seed producers have a stricter isolation requirement of 10 feet.

Production plant personnel make every effort to avoid mechanical damage to the harvested seed during the scr eening, cleaning, and bagging pr ocess. Specific methods are used to assure the genetic purity and identi ty of the seed is ma intained throughout the handling and storage operation. Bin inspections and sample collections are conducted at storage locations at the plant to examine the physical characte ristics of the soybean seed and to ensure proper bin cleanout. Seed is inspected for appearance, disease, discoloration, seed coat, mechanical damage , inert matter, and weed seed. Warm and cold germination tests are c onducted on all seed lots to ve rify acceptable germination rates. Many seed companies will also conduc t tetrazolium staining tests to assess seed viability.

Commercially certified soybean seed must m eet state and federal seed standards and labeling requirements. AOSCA standards for certified soybean seed are as follows: 98% pure seed (minimum), 2% inert matter (maxim um), 0.05% weed seed (maximum, not to exceed 10 per lb.), 0.60% total of other crop seeds (maximum), 0.5% other varieties (maximum, includes off-colored beans and off-type seeds), 0.10% other crop seeds (maximum, not to exceed three per lb.), and 80% germination and hard seed (minimum) (AOSCA, 2009a). State seed certif ication standards vary slight ly from state to state and

can be more restrictive than th e seed standards of AOSCA.

MON 87705 seed will be produc ed in the same manner as commercially certified soybean seed, such that it will meet all state and federal seed standards and labeling requirements.

IX.C. Production Management Considerations

Pre-Season

Crop rotation, tillage system, row spacing, planting equipment, seed or variety selection(s), and soil fertility require producti on decisions well in advance of planting the soybean crop. Many of the decisions in this area are made prior to or immediately after harvest of the previous crop. There are many benefits to crop rotation, with the majority of the soybean acreage planted in a two-year corn-soybean ro tation (see Section IX.G.). Crop rotation is generally a long-term decision, but the rotation sequence can be modified to take advantage of a particular economic or market opportunity. The decision to plant soybean in a conservation tilla ge or no-till system may requi re special equipment and will be made long before planting. In addition, this decision usually will be a long-term commitment, provided the system is successful . A decision to change row spacing is a similar long-term commitment that generally requires new equipment.Monsanto Company 09-SY-201U Page 145 of 471 The benefits of conservation tillage or no-till systems are well documented and include reduced soil erosion, reduced fuel and labor co sts, and conservation of soil moisture. In 2004, approximately 29.3 million acres (38.6% ) of soybean were planted in a no-till system (CTIC, 2004). Slow soybean emergence and growth plus lower yields have been

some of the concerns associated with a doption of conservation tillage systems in soybean, especially no-till. Research in Wisconsin and Minnesota shows that soil temperatures can be four to five degrees colder in no-till than conventional tillage systems, which can slow emergence, but have little effect on soyb ean yield (Pedersen, 2008a). Improved planters for establishm ent of good soybean populations and planting Roundup Ready soybean to effectively control w eeds in no-till fields have made no-till a viable production system for soybean. Res earchers still recommend some spring tillage on fine-textured and poorly drained soils for proper seedbed preparation (Pedersen, 2008a).

Most field crops, including soybean, respond well to fertilizer when pl anted in soils with low fertility levels. Soybean requires 16 essential elements for growth and development. Deficiencies in any of these elements can re duce yields (Hoeft et al., 2000). The primary or major essential nutrients are nitrogen, phosphorus and potassium. The soybean plant is a member of the legume family, like alfa lfa and clover, and fixes a significant portion of its own nitrogen through the symbiotic relationship with the nitrogen-fixing Bradyrhizobia bacteria ( Bradyrhizobium japonicum ) that live in the nodules on its roots. Bradyrhizobia are unicellar, microscopic bacteria that in vade the soybean plant through its root hairs (Hoeft et al., 2000). The plant responds to this invasion by forming nodules

which contain colonies of bact eria. Once established on the s oybean root, bacteria in the nodule take gaseous nitrogen from the atmosphere and fix it in forms easily used by the soybean plant. Since the bacteria are not na tive to U.S. soils and would not normally be found in these soils, inoculation of the s oybean seed is recommended when soybean has not been grown in a field for three to five ye ars. Nitrogen fertilizer applications at planting generally do not improve yield a nd decreases nodulation while increasing the plant’s dependency on the soil for nitrogen (Pedersen, 2008a). Therefore, nitrogen fertilizer is seldom applied prior to planting a soybean crop.

Soil tests are the only reliable way to determine the pH , phosphorus, and potassium levels in the soil. Liming and fertilizer requiremen ts subsequently are determined based on soil

test results. Ideal soil test results for corn are also ideal for soybean (Scott and Aldrich, 1970). In corn-soybean rotations in the Mi dwest, phosphorus and potassium fertilizers are applied prior to a corn crop in accordan ce with soil test recommendations, but are seldom applied prior to a soybean crop. Howe ver, in some of the southern growing areas, differences in crop rotations and soil types may require a fertilizer application prior to planting soybean.

Although not common, deficiencies can oc cur in secondary nut rients (calcium, magnesium, and sulfur) or micronutrients (b oron, chloride, copper, iron, manganese, molybdenum, and zinc). The availa bility of soil nutri ents is dependent on soil acidity or pH level. Because soybean is adversely aff ected when the pH is below approximately 5.8 (Hoeft et al., 2000), soil pH should be maintained at abou t 6.0 to 6.5 through the addition

of limestone.Monsanto Company 09-SY-201U Page 146 of 471 Soybean varieties are developed and adapte d to certain geographical zones and are separated into ten maturity groups – Group 00 to Group VIII (Pimentel, 1991; Zhang et al., 2004). Groups 00 and 0 are the earliest maturity groups a nd are adapted best to the

area north of latitude 46o north. Succeedin g groups are adapted further south with Groups I and II within latitudes 41o and 46o north, and Group III within latitudes 38o and 41o North. Group 00 through Group IV soybean va rieties are planted in the Midwest and Eastern Coastal regions. Groups II, III a nd IV account for approximately 75% (24%, 36%, and 16%, respectively) of the soybean pl anted in the U.S. (T. Schlueter, personal communication, August 2008). Groups IV through VIII are planted in the southern states with Groups V, VI and VII representing 7%, 2%, and 2% of the planted soybean, respectively (T. Schlueter, pers onal communication, August 2008).

Soybean variety selection is cr ucial for high yield and quality , and is the foundation of an effective management plan (Pedersen, 2008a). Soybean characteris tics to consider in selecting a variety include maturity, yield potential, disease and pest resistance, iron deficiency tolerance (chlorosis), lodging scor e, height, and specific soybean quality traits, such as protein and oil content. If a field has a history of a partic ular disease or pest, planting soybean varieties that ha ve resistance or tolerance to these pests and diseases can

be an effective and economical method of control.

Row spacing is important to maximize soybean yi eld. Research in the Midwest over the past 20 years consistently shows that row spac ing of less than 20 inch es is preferred for soybean regardless of tillage system, rotation sequence or planting date (Pedersen, 2008a). In the southern states , the advantage from narrow rows is less consistent and less beneficial. In 2000, approxima tely 40% of soybean was plan ted in row spacing of 10 inches or less, 27% in 10.1 to 28.5 inches, a nd 33% in rows wider than 28.5 (Hoeft et al., 2000).

Planting and Early Season

An understanding of the growth stages of soybe an is also important for the proper timing of certain management practices, such as he rbicide and insecticide applications. In addition, the impact of certai n weather conditions, insect pe sts, and diseases on soybean yield is dependent on growth st age. The system of soybean growth stages divides plant development into vegetative (V) and reproduc tive (R) stages (Pedersen, 2008a). The vegetative stages begin with VE, which designa tes emergence. V stages continue and are numbered according to how ma ny fully developed trifoliate leaves are present (i.e., V1, V2, etc.). The reproductive (R) stages begin at flowering (R1) and include pod development and plant maturation. Full maturity is designated as R8.

Adequate soil moisture and warm temperatures facilitate rapid seed germination and emergence. The ideal soil temperature for soybean germination and emergence is 77 oF (Pedersen, 2008a). However, waiting for soils to reach this soil temperature will delay planting beyond the optimum planting date th at will maximize yield. Soybean can germinate at a soil temperature of 50 oF when planted at a depth of two inches. However, emergence is slow and can take up to thre e weeks in northern climates. Because of fluctuations in soil temperature in early sp ring, soil temperature should not be the only criteria for optimum planting time. Planti ng into a good seedbed is the most importantMonsanto Company 09-SY-201U Page 147 of 471 consideration. Planting into soil that is too wet will reduce emergence and plant population, and can lead to reduced yield.

Planting date has the greatest impact on yiel d, according to research conducted in the Northern states (Hoeft et al ., 2000). Highest yields are gene rally obtained when planting is in early to mid May. Yields begin to drop off quite rapidly when planting is delayed until late May. For example, the optimum plan ting dates for soybean in Iowa are the last week of April in the southern two-thirds of the state and the first week of May in the

northern one-third of the state (Pedersen, 2008a). In the So uthern U.S., planting adapted varieties before late April results in shorter plants and, in many cases, lower yields than when the same varieties are planted in May or early June. Planting after early June generally decreases plant height and yield due to water shorta ges in July and August.

Variations in plant spacing through row spacing and plant population have a significant effect on canopy development and soybean yiel d. Soybean has the ability to produce good yield over a wide range of plant populati ons. Most soybean varieties have the ability to branch and adjust the number of pods on branches to compensate for large differences in seeding rate. Maximum yields generally require planti ng rates that result in about 2.5 to 5 plants per square foot (H oeft et al., 2000). Ther efore, a full stand of soybean is approximately eight to ten plants per foot of row at ha rvest for 40-inch rows, six to eight plants per foot of row in 30-inch rows, four to six plants in 20-inch rows, and two to three plants in 10-inch rows. This translates to 109,000 to 218,000 plants per acre at harvest. Higher populations are recomm ended in narrow rows for maximum yields because plants are more uniformly spaced in na rrow rows. Seeding rates are generally 10 to 25% higher than the desired harvest populatio n, especially in no-till, to account for the losses in germination, emergence, and seed ling diseases. The accuracy of the planting equipment also can impact the decision on seed ing rate. Soybean se ed traditionally has been sold by weight. Therefore, the farm er must know the number of seeds per pound for the particular soybean varieties being planted for accurate seeding rates.

Treating soybean seed with a fungicide (e.g., pyraclostrob in, metalaxyl, or mefenoxam) to prevent damping-off diseases may be beneficial when plan ting in cold, wet soils, using reduced till and no-till planting systems, and when planting seed with a low germination rate (<80%) or low seed vigor.

Annual and perennial weeds are considered to be the greates t pest problem in soybean production (Aref and Pike, 1998). In order to maximize yields , weeds must be controlled during the early growth stages of soybean because weeds compete with soybean for water, nutrients, and light. A combination of tillage and herbicides are used to control weeds throughout the growing season.

Mid to Late Season

Ideal daytime temperatures for soybean growth are between 75 oF and 85 oF (Hoeft et al., 2000). Warmer temperatures result in larger plants and earlier fl owering. Sustained temperatures below 75 oF will delay the beginning of flowering significantly. Seed set also is affected by temperature. Seed set is generally good when pollination follows night temperatures around 70 oF. Soybean vari eties differ in their response and tolerance

to temperatures.Monsanto Company 09-SY-201U Page 148 of 471 Soybean is photoperiod sensitive, which mean s that it transitions from vegetative to flowering stage in direct res ponse to length of daylight (S cott and Aldrich, 1970). Most soybean varieties begin floweri ng soon after the day length begi ns to shorten. Flowering of southern varieties is initiated by a shorte r day than that of varieties adapted to the north. The extent of vegetative growth occu rring after the initiation of flowering depends not only on environmental factors but also th e growth habit. Soybean varieties are

described as either indeterminate or determin ate in their growth habit (Scott and Aldrich, 1970). Indeterminate varieties in crease their height by two to four times after flowering begins. These are grown in the northern and central U.S. Determinate varieties increase their height very little after flowering and generally are grown in the southern U.S. Indeterminate and determinate varieties also differ in flowering characteristics. Indeterminate plants generall y bloom first at the fourth or fifth node and progress upward. Flowering on determin ate plants begins at the eight or tenth node and progresses both downward and upward.

The first appearance of flow ers signals the beginning of th e reproductive stage, namely the R1 stage (Hoeft et al., 2000). The reprodu ctive period consists of flowering, pod set,

and seed formation. Climatic conditions su ch as temperature and moisture supply during the flowering period will affect the number of flowers. Th e soybean plant does not form a pod for each flower. It is common for the s oybean plant to have 75% of the flowers fail to develop a pod (Scott and Aldrich, 1970). This characteristic makes soybean less susceptible than corn to s hort periods of adverse weathe r during flowering. Under normal conditions, pod set occurs over about a three week period. Good soil moisture is most critical during the pod-f illing stages to prevent pod a bortion and to ensure high yields (Hoeft et al., 2000). Another critical period is during the seed-f illing stages to assure high rates of photosynthesis. Hi gh humidity and temperatures during seed development and maturity can result in poor seed quality because these conditions promote the development of re productive-stage diseases.

Harvest Season

When dry matter accumulation ends , the plant is considered to be physiologically mature. The seed moisture content is approximately 55 to 60% at this stage (Hoeft et al., 2000). At this stage, namely R7, at least one normal pod on the plant reaches the mature pod color. Under warm and dry w eather conditions, seed moisture content will drop to 13 to 14% in 10 to 14 days from physiological maturity (Hoeft et al., 2000). Soybean can be harvested when the moisture content drops be low 15%. However, s oybean should be at

13% moisture to be stored without artificia l drying (Scott and Aldrich, 1970). Moisture content below 12% may increase seed cracking and seed coat damage.

Pre-harvest losses are influenced by variety, weather, and timeliness of harvest (Scott and Aldrich, 1970). Timely harvest when the mo isture content is 13 to 14% also will minimize losses. Proper operation and adjust ment of the combine is essential to minimizing harvest losses in the field.Monsanto Company 09-SY-201U Page 149 of 471 IX.D. Weed Management

Annual weeds are perceived to be the grea test pest problem in soybean production, followed by perennial weeds (Aref and Pike, 1998). Soybean insects and diseases are rated less problematic but may reach economic thresholds requirin g treatment. Weed control in soybean is essential to optimizing yields. Weeds compete with soybean for light, nutrients, and soil moisture. Weeds can harbor insects and di seases, and also can interfere with harvest, causi ng extra wear on harvest equi pment (Pedersen, 2008a). The primary factors affecting soybean yield loss fr om weed competition are the weed species, weed density, and the duration of the competitio n. When weeds are left to compete with soybean for the entire growing season, yield lo sses can exceed 75% (Dalley et al., 2001). Generally, the competition increases with increasing weed density. The time period that weeds compete with the soybean crop influences the level of yield loss. In general, the later the weeds emerge, the less impact the weeds will have on yield. Soybean plants withstand early-season weed competition longer than corn, and the canopy closes earlier in soybean than corn. In addition, canopy clos ure is much sooner when soybean is drilled or planted in narrow rows. The most common weeds in soybean for each of the three major growing regions are presented in Tables IX-4, IX-5 and IX-6.

Crop rotations and environment have a significant impact on the adaptation and occurrence of weeds in soybean. Foxta il spp. (foxtail species group), pigweed, velvetleaf, lambs quarters, and cocklebur are common weeds in Midwest corn and soybean fields. However, growers consider giant ragweed ( Ambrosia artemisiifolia ), lambs quarters ( Chenopodium album ), Canada thistle ( Cirsium arvense ), cocklebur ( Xanthium strumarium ), and velvetleaf ( Abutilon theophrasti ) to be the top five most problematic weeds in corn and soybean because of the difficulty to control these weeds (Nice and Johnson, 2005). The most frequen tly reported common weeds in the Southeast region are morning glory ( Ipomoea spp .), prickly sida (Sida spinosa), johnsongrass ( Sorghum halepense ), sicklepod ( Cassia obtusifolia ), and broadleaf signalgrass ( Brachiaria platyphylla ) (Webster et al., 2005).Monsanto Company 09-SY-201U Page 150 of 471 Table IX-4. Common Weeds in Soybean Production: Midwest Region

Foxtail spp. (12) 1 Ragweed, giant (3) Dandelion (1) Pigweed spp. (11) Shatte rcane (3) Johnson grass (1) Velvetleaf (11) Quackgrass (3) Milkweed, honeyvine (1) Lambsquarters (10) Buckwheat, w ild (2) Nightshade, hairy (1) Cocklebur (9) Crabgrass spp. (2) Oats, wild (1) Ragweed, common (7) Kochia (2) Pokeweed, common (1) Smartweed spp. (6) Mustard, wild (2) Prickly sida (1) Morning glory spp. (5) Nightshade, Eastern black (2) Proso millet, wild (1) Sunflower, spp. (5) Palmer amaranth (2) Sandbur, field (1) Waterhemp spp. (5) Canada thistle (1) Venice mallow (1) Horseweed (marestail) (3) Chickw eed (1) Volunteer cereal (1) Panicum, fall (3) Cupgrass, w oolly (1) Volunteer corn (1)

1 Number provided in parenthesis is the number of states out of the thirteen total states in the Midwest region reporting each weed as a common weed.

Sources: IL: University of Illinois (2002) and Aaron Hage r, Extension Weed Specialist, University of Illinois - Personal Communication (2006).

IN: 2003-2005 Statewide Purdue Horseweed Weed Survey, Special database query and personal communication (2006), Bill Johnson, Extension Weed Specialist, Purdue University. IA, MN, OH, WI: WSSA, 1992. KS: Dallas Perterson, Extension Weed Specialis t, Kansas State - Personal communication (2006). KY, MO: Webster et al., 2005. MI: Davis, et al., 2005. List is not ranked in order of importance or frequency. NE: Alex Martin, Extension Weed Specialist, Un iversity of Nebraska – Personal communication (2006). ND: Zollinger, 2000. SD: Michael Moechnig, Extension Weed Speci alist, South Dakota State University – Personal communication (2006).

Table IX-5. Common Weeds in Soybean Production: Mid-South Region

Morning glory spp. (5) 1 Pigweed spp. (3) Ragweed, common (1) Prickly sida (5) Crabgrass spp. (2) Ragweed, giant (1) Johnson grass (4) Palmer amaranth (2) Red rice (1) Sicklepod (4) Cocklebur (1) Smartweed (1) Signalgrass, broadleaf (4) Copperleaf , hophorn (1) Spurge, nodding/hyssop (1) Barnyard grass (3) Florida pus ely (1) Spurge, Prostrate (1) Hemp sesbania (3) Horseweed (marestail) (1) Nutsedge spp. (3) Poinsettia, wild (1)

1 Number provided in parenthesis is the number of states out of the five total states in the MidSouth region reporting each weed as a common weed.

Sources: AL, LA, MS, TN: Webster et al., 2005. AR: Ken Smith, Extension Weed Specialist, Un iversity of Arkansas - Personal communication (2006).Monsanto Company 09-SY-201U Page 151 of 471 Table IX-6. Common Weeds in Soybean Production: Eastern Coastal Region

Ragweed, common (8) 1 Jimson weed (4) Dandelion (1) Cocklebur (7) Sicklepod (3) Goosegrass (1) Morning glory spp. (7) Florida pusel y (2) Nightshade, Eastern black (1) Crabgrass spp. (6) Johnson grass (2) Panicum, Texas (1) Foxtail spp. (6) Palmer amar anth (2) Prickly sida (1) Lambsquarters (6) Quackgrass (2) Shattercane (1) Pigweed spp. (6) Arrowleaf sida (1) Signalgrass, broadleaf (1) Velvetleaf (6) Beggarweed, Flor ida (1) Smartweed spp. (1) Nutsedge spp. (5) Burcucumber (1) Panicum, fall (5) Canada thistle (1)

1 Number provided in parenthesis is the number of states out of the eight total states in the Eastern Coastal region reporting each weed as a common weed. Data were not available for DE in soybean.

Sources: DE, MD, NJ, PA: WSSA, 1992. GA, NC, SC: Webster et al., 2005. NY: Russell Hahn, Extension Weed Specialist, Cornell University – Personal Communication (2006). VA: Scott Hagood, Extension Weed Specialist, Virginia Tech – Personal Communication (2006).

Cultural and mechanical weed control practices are important components of an effective weed management program (Baumann et al ., 2008). Crop rotation, narrow row spacing and planting date are a few of the crop management practices that are implemented to provide the crop with a competitive edge ove r weeds. Although the primary purpose of tillage is for seedbed preparation, tillage is still used to supplement weed control with selective herbicides in soybean production. Approximately 98% of the soybean acreage

received an herbicid e application in 2006 indicating th e importance of excellent weed control in maximizing soybean yield (USDA-ERS, 2007). Herbicide-tolerant soybean were introduced to provide grow ers with additional options to improve crop safety and/or improve weed control. The Roundup Ready soybean sy stem, i.e., planting Roundup Ready soybean and applying glyphosate in crop , has become the standard weed control program in U.S. soybean production and is planted on 92% of the soybean acreage (USDA-NASS, 2008).

Herbicides provide effective and economical co ntrol of weeds in soybean. The risk of weeds developing resistance to herbicides and the potential impact of resistance on the usefulness of a herbicide vary greatly acro ss different mechanisms of action and are dependent on a combination of factors, such as selection pressure, herbicide soil residual activity, herbicide chemistry, prolific s eed production and high genetic variation in plants. Weed-resistance management programs th at integrate the use of herbicides with

different mechanisms of acti on and short residual activity times in soil reduce selection pressure (Prather et al., 2000). In conjunc tion with crop rotation, which may allow the grower to manipulate planting times to avoi d early-season weed ge rmination (Jordan et al., 1995) and to use mechanical as well as chemical weed control methods, these practices impede the development of herbicid e resistance in weeds. Monsanto investsMonsanto Company 09-SY-201U Page 152 of 471 considerably in resear ch to understand the proper uses and stewardship of the glyphosate

molecule. This research include s an evaluation of some of th e factors that can contribute to the development of weed resistance. De tailed information regarding glyphosate weed resistance management is presented in Appendix L.

IX.E. Management of Insects

Although insects are rated as le ss problematic than weeds in U.S. soybean production, management of insect pests during the growth and development of soybean is important for protecting the yield of s oybean (Aref and Pike, 1998). Understanding the impact of insects on soybean growth is essential fo r proper management (Higley and Boethel, 1994). It is important to unders tand the way that insects inju re soybean as well as how the soybean plant responds to insect injury. Insect inju ry can impact yield, plant maturity, and seed quality. Injury is define d as a stimulus producing an abnormal change in plant physiological processes. Injury ma y produce stress, whic h is a departure from optimal physiological conditions (Higley and Boethel, 1994). The ultimate impact of injury is damage: a measurable reduction in plant growth developm ent or reproduction. Insect injury in soybean seldom reaches levels to cause an economic loss, as indicated by the low percentage (16%) of soybean acreage that receives an insecticide treatment (USDA-NASS, 2007).

Characterizing soybean responses to insect injury is esse ntial in establishing economic injury levels (Higley and Boet hel, 1994). Most often, soybe an insects are categorized or defined by the plant parts they injure, namely root-feeding, st em-feeding, leaf-feeding, or pod-feeding insects. The root- and stem-feedi ng insect groups are often the hardest to scout and typically are not det ected until after they have cau sed their damage. The leaffeeding insects comprise the biggest group of insects, but not necessarily the most damaging insects. Recent research on defolia tion has determined that a major effect of injury is to reduce light interception by th e soybean canopy which in turn can have a significant effect on yield (H igley and Boethel, 1994). S oybean has an extraordinary capacity to withstand consider able defoliation early in the season without significant yield loss. By contrast, defoliation during the flowering and pod filling stages poses a greater threat to yield because the soybean pl ant has less time to compensate for injury compared to other growth stages. Research indicates that the soybean plant can sustain a 35% leaf loss prior to the pre-bloom period w ithout lowering yiel d (NDSU, 2002). However, from pod-set to maturity, the plan t can tolerate only a 20% defoliation level before yield is impacted.

IX.F. Management of Di seases and Other Pests

More than 100 pathogens are known to affect soybean, of which 35 are considered to be of economic importance (Heathe rly and Hodges, 1999). The estimated yield losses to soybean diseases in the U.S. were 10.9, 11.9, and 14.0 million metric tons in 1996, 1997, and 1998, respectively (Wrather et al., 2000). Pathogens can affect all parts of the soybean plant, resulting in reduced quality a nd yield. The extent of losses depends upon the pathogen, the state of plant developmen t and health when infection occurs, theMonsanto Company 09-SY-201U Page 153 of 471 severity of the disease on individual plants , and the number of plan ts affected (Heatherly and Hodges, 1999).

One or more diseases can generally be f ound in fields wherever soybean is grown (Heatherly and Hodges, 1999). However, a path ogen may be very destructive one season and difficult or impossible to find the next s eason. The extent and severity of soybean diseases depend on the degree of compatibility between the host and the pathogen and the

influence of the environment.

According to field surveys conducted in fift een soybean-producing st ates during 1996 to 1998, soybean cyst nematode ( Heterodera gylcines ) caused the greatest soybean yield losses (Wrather et al., 2000). Ph ytophthora root and stem rot ( Phytophthora sojae ), brown stem rot ( Phialophora gregata ), sclerotinia stem rot ( Sclerotinia sclerotiorum ), and seedling diseases followed in economical importance. As expected, yield losses varied by region. Sclerotinia stem rot caused yield losses in several Northern states, but

not in other states. Rhizoctoni a foliar blight losses were grea test in Arkansas, Louisiana, and Texas where humidity and temperatur e conditions are suitable for disease development.

Selecting resistant varieties is the primar y tool growers have for disease control (Heatherly and Hodges, 1999). Resistant varieties may have morphological or physiological characteristics that provide immunity, resistance, toleran ce or avoidance to certain pathogens. Cultural practices play an important role in disease management by reducing initial inoculums or reducing the rate of diseas e development (Heatherly and Hodges, 1999). Preplant tillage can bur y crop residue, which encourages the decomposition of fungal-resting structures. Crop rotation is routinely recommended as a disease-management strategy. Rotating crops interrupts the disease cycle and allows time for the decomposition of inoculums. One exception is Rhizontonia, a soil-inhabitant pathogen that grows on a wide va riety of crops and can survive sufficiently in the soil to make crop rotation as a means of controlling this pest impractical. Row spacing, plant population, and planting date also can be changed to manage soybean diseases.

Soybean cyst nematode (SCN ) is one of the most dama ging pathogens of soybean throughout the soybean growing regions of the U.S. (Pedersen, 2008b). Losses have
been estimated to be at about $1.5 billion in the U.S. (Pedersen, 2008a). SCN can cause yield losses up to 50%, where this pest in 2004 alone caused an estimated loss of 50 million bushels in Iowa (Pedersen, 2008b). So ybean cyst nematodes feed on the roots, causing severely stunted and yellow plants. The simplest, least expensive method to reduce populations of this pest is to rotate s oybean with a non-host crop such as corn, small grains, or sorghum. Planting resistant va rieties is regarded as the best and most effective management practice to prevent lo sses from this pest. Several public and private soybean varieties offer sources of resistance to certain races of nematode. Alternating varieties with di fferent sources of resistan ce also is beneficial.

High-quality seed is essential for control ling seedling diseases. The most important seedling diseases in soybean are Phytophthora, Pythium, Rhizoctonia, and Fusarium (Pedersen, 2008a). Many soybean varieties demonstrate resistance to specific taxonomic races of Phytophthora. Treati ng soybean seed with a fungicide (e.g., pyraclostrobin, metalaxyl, mefenoxam) is effective against da mping-off disease (seedling blight) causedMonsanto Company 09-SY-201U Page 154 of 471 by common soil fungi, such as P hytophthora and Pythium. Fungi cide seed treatments are recommended where there is a history of these seedling diseases.

Asian soybean rust is a foliar fungal dise ase that typically infests soybean during reproductive stages of development and can cause defoliation and reduce yields significantly in geographies su ch as Brazil (Dorrance et al., 2007). Soybean rust is caused by the fungus Phakopsora pachyrhizi . This disease in the U.S. was first detected

in Louisiana in 2004. At th is time, foliar application of fungicides is the standard disease-management practice to limit yield losses due to soybean rust.

Foliar fungicide applications can effectively reduce the incidence of many diseases (Heatherly and Hodges, 1999). However, the economic return from a fungicide application may be limited to select pr oduction programs; for example, high-yield environments or when producing soybean s eed. According to USDA-NASS statistics, fungicides were applied on approximately 4% of the soybean acreage in 2006 (2007).

IX.G. Crop Rotation Practices in Soybean

The well-established farming practice of crop ro tation is still a key management tool for growers. The purposes of growing soybean in rotation are to improve yield and profitability of one or both crops over ti me, decrease the need for nitrogen fertilizer on the crop following soybean, increase residue co ver, mitigate or break disease, insect, and weed cycles, reduce soil erosion, increase soil organic matter, improve soil tilth and soil physical properties, and reduce runoff of nutrients, herbicides, and insecticides (Boerma and Specht, 2004; Al-Kaisi et al., 2003). According to USDA Economic Research Service, 95% of the soybean-planted acreage has been in some form of a crop rotation system since 1991 (USDA-ERS, 2001). Co rn- and wheat-planted acreage has been rotated at a slightly lower level of 75% and 70%, respectively. A lthough the benefits of crop rotations can be substant ial, the grower must make cropping decisions by evaluating both the agronomic and economic returns on various cropping systems. Crop rotations also afford growers the opportu nity to diversify farm produc tion in order to minimize

market risks.

Continuous soybean production is not a co mmon practice in the Midwest and is discouraged by most extension soybean specialists to reduce the risk of diseases and

nematodes (Hoeft et al., 2000; Al-K aisi et al., 2003). Corn and soybean occupy more than 80% of the farmland in many of the Mi dwestern states, and the two-year cropping sequence of soybean-corn is used most extensively in this region. However, a soybean crop sometimes is grown after soybean and th en rotated to corn in a 3-year rotation sequence (soybean-soybean-corn) in the Midwest. Compared to corn, soybean shows a greater yield response to bei ng grown after a number of year s without soybean. The yield of both corn and soybean is approximately 10% higher when grown in rotation than when either crop is grown continuous ly (Hoeft et al., 2000).

A combination of conservation tillage practi ces and crop rotation has been shown to be very effective in improving soil physical prope rties. Long-term studies in the Midwest indicate that the corn-soybean rotation im proves yield potential of no-till systems compared to continuous corn production (Al- Kaisi, 2001). The reduction in yield ofMonsanto Company 09-SY-201U Page 155 of 471 continuous corn production in no-till systems is attributed to low so il temperature during seed germination, which is evident on poor ly drained soils unde r no-till practices.

Crop rotations may change over a long period of time due to economic conditions and market opportunities. Roundup Ready soyb ean has provided growers more profit opportunities than conv entional soybean primarily by reducing input costs (Gianessi, 2005). In addition, Roundup Ready soybean ha s provided growers greater flexibility to grow soybean in fields with weed infestations, which previ ously were considered to be too problematic or unproductive for growing.

Unique to the southern portion of the Midw est and the Mid-South regions, soybean is grown in a double-cropping system. Double-cr opping refers to the practice of growing two crops in one year. This practice can improve income and reduce soil and water losses by having the soil covered with a plan t canopy most of the year (Hoeft et al., 2000). In the Midwest, winter wh eat is harvested in late June or July, and then soybean is planted into the wheat residue in a no-till system to conserve moisture. Due to the uncertainty of double-cropping yiel ds, growers sometimes do not plant if soils are too dry at the time of wheat harvest. Soybean t ypically is grown in a corn-wheat-soybean rotation sequence when soybean is grown in a double-croppi ng system. In the northern soybean growing areas, wheat will typically follow soybean in the rotation.

Agronomic practices such as rotations for soyb ean vary from state to state. However, there are similarities among states within cert ain growing regions. This section provides a detailed description and qua ntitative assessment of the rotational cropping practices immediately following soybean pr oduction, by state. This assessment accounts for about 99% of the total soybean acreage. These data are presented in Tabl es IX-7 through IX10.

The majority of the U.S. soybean acreage (68. 6%) is rotated to corn (Table IX-7). The second largest rotational crop following soybean is soybean. Approximately 14.5% of the soybean acreage is rotate d back to soybean the follo wing year. Wheat follows soybean on approximately 11.2% of the U.S. soybean acreage, with cotton, rice, and sorghum the next largest rotational crops fo llowing soybean. However, these three crops were planted on only 4.6% of the soybean ac reage. Other minor rotational crops that follow soybean production are listed in Tables IX-7 through IX-10.

Column J of each table provide s the percentage of soybean acreage as a function of the total rotational crop acreage to indicate the level that soybean is the primary crop preceding the rotational crops. For the entire c ountry (Table IX-7), this percentage is 35.3% indicating that soybean is a major crop preceding these rotational crops. The percentage of soybean as a preceding crop varies widely in different states, which ranges from 16.8% (KS) to 95.2% (NJ). In the Midwest region where 82% of the soybean is grown, 34.6% of the rotational crop area wa s planted with soybean during the previous growing season.

One rotation choice available to growers is to plant another Roundup Ready crop following the production of glyphosate-tolera nt soybean. To de termine the likelihood that the rotational crops planted after MON 87705 will be another Roundup Ready crop, an assessment also has been provided in Tabl es IX-7 through IX-10. This assessment is based on current agronomic practices fo llowing soybean production. Roundup ReadyMonsanto Company 09-SY-201U Page 156 of 471 alfalfa, canola, corn, cotton, soybean, and sugar beets have been nonregulated by the USDA and were considered as potent ial Roundup Ready crops following soybean production. For the purposes of this asse ssment, the adoption rates used for Roundup Ready corn, cotton, and soybean in 2008 we re obtained from the USDA-NASS Acreage Summary report (2008c). The percentages for Roundup Ready corn, cotton, and soybean in the following tables were assumed to be the total percentage of herbicide-tolerant crops because the USDA-NASS report does not show the percentages of each individual

herbicide-tolerant trait. Therefore, this is a slight overstatement for Roundup Ready corn, cotton, and soybean since other herbicide-to lerant traits are pl anted. Roundup Ready sugar beets were commercially introduced in 2008, although the USDA-NASS report does not provide planting information for this biotechnology-derived crop. Therefore, a 90% adoption rate will be assumed for fu ture plantings. Considering Roundup Ready alfalfa was nonregulated by the USDA, but is currently not availabl e in the marketplace, a future market adoption estimate will be used. An adoption rate of 50% will be assumed for Roundup Ready alfalfa.

This assessment showed that the percentage of the total rotational crop acreage that may be rotated from Roundup Ready soybean to another Roundup Ready crop (Table IX-7 - Column K) is estimated to be 21.0% in the U.S. and ranges from 7.1% (KS) to 78.3% (MS) across the soybean growing states. The percentage is 20.0% in the Midwest region, which is the largest s oybean growing region.Monsanto Company 09-SY-201UPage 157 of 471Table IX-7. Rotational Practices in the U.S. Following Soybean ProductionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop in theU.S.1% RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated% RoundupReadyCrops asMajorRotations8UnitedStates75037CornSoybeanSorghumCottonWheatBarleyOatsRiceAlfalfaSugar BeetsPotatoesDry BeansDry PeasMilletFlaxOther98013075037402037673741421591995230118648303341183520250345452Total: 21260164.314.520.941.722.41.94.945.38.717.39.63.07.316.422.034.3515001086684115708396419810421621443235384176155Total: 7503768.614.51.12.111.20.050.11.40.20.190.040.050.050.050.100.264.592.6NA79.3NANANANA5090NANANANANANA3321410062124581129Total: 4473135.321.0The U.S. summary (Table IX-7) was developed by compiling the data from all three regional summaries. NA denotes not applicable. All acreages are expressed as1000s of acres.1Acreage planted of the specific crops is based on 2008 plantingdata (USDA-NASS, 2008); “other” crop and newly seeded alfalfaacreages are based on 2008 plantingdata from the Individual States data which was obtained from Quick Stat searcheson http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E is obtained by dividing Column F by Column D.3Column F is obtained by multiplying Column B by Column G.4Column G is obtained by dividing Column F by Column B.5Column H is obtained by dividing Column I by Column F.6Column I is obtained by compiling the data from all three regional summaries.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column I Total by Column D Total.9Various vegetables.Monsanto Company 09-SY-201UPage 158 of 471Table IX-8. Rotational Practices FollowingSoybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %RoundupReady Cropsas MajorRotations8Region 62150CornSoybeanSorghumCottonWheatBarleyOatsRiceAlfalfa9Sugar BeetsPotatoesDry BeansDry PeasMilletFlaxOther1072260621503553341320391929159020016178302781166520250345342Total: 17941065.77.918.822.925.32.16.291.010.017.311.63.07.316.422.025.34748048856707881024198182162144323538417687Total: 6215076.47.91.10.113.00.070.20.30.30.20.050.060.060.070.10.165.492.6NA87.2NANANANA5090NANANANANANA3104045236881129Total: 3584134.620.0IL 9200CornSoybeanSorghumWheat121009200801200Total: 225807139228855623074340Total: 920093.02.50.83.767%87%NANA5733200Total: 593340.726.3IN 5450CornSoybeanWheat57005450580Total: 11730865474905273273Total: 5450905571%96%NA3483262Total: 374446.531.9Monsanto Company 09-SY-201UPage 159 of 471Table IX-8 (cont.). Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCrop Option5Acreage ofRoundupReadyRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %RoundupReady Cropsas MajorRotations8IA 9750CornSoybeanAlfalfa9133009750125Total: 2317571278945819598Total: 97509721689550643118549Total: 666542.128.8KS 3300CornSoybeanSorghumWheat3850330029009600Total: 19650431061216503301651155Total: 330050105356595NANA1073314Total: 138616.87.1KY 1390CornSoybeanWheat12101390580Total: 3180981012118213970Total: 1390851055487NA638121Total: 75943.723.9MI 1900CornSoybeanWheat24001900730Total: 503055565133095475Total: 1900705255784NA75880Total: 83837.816.7MN 7050CornSoybeanWheatSugar beetsDry BeansOther11770070501925440150203Total: 1746870366242435535821212691063571Total: 7050763181.50.516991NA90NANA369719295Total: 398440.422.8Monsanto Company 09-SY-201UPage 160 of 471Table IX-8 (cont.). Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %RoundupReady Cropsas MajorRotations8MO 5200CornSoybeanSorghumCottonWheatRice28005200903061250200Total: 984698301162542912756156010478520182Total: 5200533021.5103.54392NA87NANA1185143568Total: 268852.827.3NE 4900CornSoybeanSorghumWheat880049003001750Total: 15750421082283675490245490Total: 490075105105997NANA2168475Total: 264431.116.8ND 3800CornSoybeanWheatSugar BeetsDry PeasFlax255038009230208520335Total: 166434521191872311407981710383876Total: 38003021451126594NA90NANA74175034Total: 152522.89.2OH 4500CornSoybeanWheat330045001120Total: 89209510803150450900Total: 45007010205489NA1701401Total: 210250.423.6Monsanto Company 09-SY-201UPage 161 of 471Table IX-8 (cont.). Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %RoundupReady Cropsas MajorRotations8SD 4100CornSoybeanSorghumWheatBarleyOatsMillet47504100170366163220110Total: 13074622482265373729528282820418241Total: 41007222201218897NANANANANA259880Total: 267731.420.5WI 1610CornSoybeanWheatOatsAlfalfa9PotatoesOther123800161037327042063.5139.2Total: 66763622261551121369328116643216Total: 1610852514216190NANA50NANA8352932Total: 89624.113.4The Midwest region summary (Table IX-8) was developed by compiling the data from all the states within the region. Unlike theindividual state data, the datain Column G for this regionalsummary were obtained by dividing Column F byColumn B and the data in Column H wereobtained by dividing Column I by Column F.NA denotes not applicable. All acreages areexpressed as 1000s of acres.1Acreage planted of the specific crops is based on 2008 plantingdata (USDA-NASS, 2008); “other”crop and newly seeded alfalfaacreages are based on 2008 planting data from theIndividual States data which were obtained from Quick Statsearches on http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E is obtained by dividing Column F by Column D.3Column F is obtained by multiplying Column B by Column G.4The rotational crop percentages are based on estimates from personal communications(2006) with individual state Extension Crop Production Specialist; Extension Agronomists –Soybean, Corn and Cotton; Extension Weed Control Specialist on Soybean and Corn; and/or Monsanto Technology Development Representatives.5Roundup Ready rotational crop adoption rates for corn, soybean and cotton are based on 2008 planting data (USDA-NASS, 2009a). The percentages for Roundup Ready corn, cotton andsoybean represent the percentagesfor total herbicide-tolerant traits. Percentages of herbicide-tolerant alfalfa and sugar beets are future market adoption estimates.6Column I is obtained by compiling the data from all the states within the region.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column I Total by Column D Total.9Newly seeded alfalfa.10Various vegetables.11Sweet corn and green peas.12Sweet corn, green peas and onions.Monsanto Company 09-SY-201UPage 162 of 471Table IX-9. Rotational Practices Following Soybean Production in the Mid-South RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4%RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated% RoundupReadyCrops asMajorRotations8Region 8200CornSoybeanSorghumCottonWheatRice26308200368186028502101Total: 1800943.163.046.532.69.440.911325164171606267860Total: 820013.863.02.17.43.310.554.093.3NA82.8NANA6124820502Total: 593445.532.9AL 360CornSoybeanCottonWheat260360290240Total: 115048562151261818036Total: 3603555010548780NA6816144Total: 22831.319.8AR 3300CornSoybeanSorghumWheatRice440330012510701401Total: 63365364532247231211266231660Total: 330076427205494NANANA1251985Total: 211052.133.3LA 1050CornSoybeanSorghumCotton5201050120300Total: 199020658853105683105158Total: 1050106510155487NA7957594124Total: 77552.838.9Monsanto Company 09-SY-201UPage 163 of 471Table IX-9 (continued). Rotational Practices Following Soybean Production in the Mid-South RegionA B C D E F G H I J KStateTotalSoybeanAcres1MajorCropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated% RoundupReadyCrops asMajorRotations8MS 2000SoybeanRice2000230Total: 223090871800200Total: 2000901097NA1746Total: 174689.778.3TN 1490CornSoybeanCotton6901490285Total: 2465973794671551268Total: 1490453718548787362480233Total: 107560.443.6The Mid-South region summary (Table IX-9)was developed by compiling the data from all the states within the region. Unlike the individual state data, the data inColumn G for this regional summary were obtained by dividing ColumnF by Column B and the data in Column H were obtained by dividing Column I by Column F.NA denotes not applicable. All acreages are expressed as 1000s of acres.1Acreage planted of the specific crops is based on 2008 plantingdata (USDA-NASS, 2008); “other” crop and newly seeded alfalfaacreages are based on 2008 plantingdata from the Individual States data which were obtained from Quick Stat searcheson http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E is obtained by dividing Column F by Column D.3Column F is obtained by multiplying Column B by Column G.4The rotational crop percentages are basedon estimates from personal communications (2006) with individual state Extension Crop Production Specialist; ExtensionAgronomists – Soybean, Corn and Cotton; Extension Weed Control Specialist on Soybean and Corn ;and/or Monsanto Technology Development Representatives.5Roundup Ready rotational crop adoption rates for corn, soybean and cotton are based on 2008 planting data (USDA-NASS, 2009a).The percentages for RoundupReady corn, cotton and soybean represent the percentages for totalherbicide-tolerant traits. Percentages of herbicide-tolerant alfalfa and sugar beets are future marketadoption estimates.6Column I is obtained by compiling the data from all the states within the region.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column I Total by Column D Total.Monsanto Company 09-SY-201UPage 164 of 471Table IX-10. Rotational Practices Following Soybean Production in the Eastern Coastal RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1% RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %RoundupReady Cropsas MajorRotations8Region 4687CornSoybeanCottonWheatOther95240468715662525110Total: 1412855.217.656.61.149.528928278872754Total: 468761.717.618.90.61.254.087.076.1NANA1562719675Total: 295633.220.9DE 195CornSoybean160195Total: 355982015639Total: 195802054878434Total: 11554.933.3GA 430CornCotton370940Total: 1310124143387Total: 4301090547823302Total: 32532.824.8MD 495CornSoybean460495Total: 955971044650Total: 4959010548724143Total: 28451.829.7NJ 92CornOther108511.6Total: 979779839Total: 92901054NA45Total: 4595.246.3NY 230CornOther11109033.5Total: 1124203421912Total: 23095554NA118Total: 11820.510.5NC 1690CornSoybeanCottonOther12900169043065Total: 30859025985281142342334Total: 16904825252548776NA438368321Total: 112754.836.5Monsanto Company 09-SY-201UPage 165 of 471Table IX-10 (continued). Rotational Practices FollowingSoybean Production in the Eastern Coastal RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1%RotationalCropFollowingSoybean2RotationalCrop AcresFollowingSoybean3%RotationalCrop ofTotalSoybean4% RoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %RoundupReady Cropsas MajorRotations8PA 435CornSoybean1350435Total: 17853224269Total: 43598254872308Total: 23824.413.3SC 540CornSoybeanCottonWheat355540135220Total: 1250843040122971625427Total: 5405530105548768NA16014137Total: 33843.227.0VA 580CornSoybeanCotton47058061Total: 111188253841214523Total: 5807125454876822212616Total: 36452.232.8The Eastern Coastal region summary (Table IX-10) was developed by compiling the data from all the states within the region. Unlike the individual state data, the datain Column G for this regional summary were obtained by dividing Column F by Column B and the data in Column H were obtained bydividing Column I by Column F.NA denotes not applicable. All acreages are expressed as 1000s of acres.1Acreage planted of the specific crops is based on 2008 plantingdata (USDA-NASS, 2008); “other” crop and newly seeded alfalfaacreages are based on 2008 plantingdata from the Individual States data which were obtained from Quick Stat searcheson http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E is obtained by dividing Column F by Column D.3Column F is obtained by multiplying Column B by Column G.4The rotational crop percentages are basedon estimates from personal communications (2006) with individual state Extension Crop Production Specialist; ExtensionAgronomists – Soybean, Corn and Cotton; Extension Weed Control Specialist on Soybean and Corn; and/or Monsanto Technology Development Representatives.5Roundup Ready rotational crop adoption rates for corn, soybean and cotton are based on 2008 planting data (USDA-NASS, 2009a).The percentages for RoundupReady corn, cotton and soybean represent the percentages for totalherbicide-tolerant traits. Percentages of herbicide-tolerant alfalfa and sugar beets are future marketadoption estimates.6Column I is obtained by compiling the data from all the states within the region.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column I Total by Column D Total.9Sweet corn and other vegetables.10Sweet corn, and other vegetables.11Sweet corn and onions.12Cucumbers and sweet potatoes.Monsanto Company 09-SY-201U Page 166 of 471 IX.H. Soybean Volunteer Management

Volunteer soybean is defined as a plant that has germinated and emerged unintentionally in a subsequent crop. Soybean seeds can re main in a field after soybean harvest as a result of pods splitting before or during harves t. Soybean seeds also can remain in a field when pod placement on the plants is too clos e to the ground for the combine head to collect all the pods or when the combine is im properly adjusted for efficient harvesting. Volunteer soybean in rotationa l crops is not a concern in the Midwest region because the soybean seed is typically not viable after the winter period (C arpenter et al., 2002; OECD, 2000). In southern soybean growing areas of th e U.S. where the winter temperatures are milder, it is possible for soybe an seed to remain viable over the winter and germinate the following spring.

Volunteer soybean normally is not a concern in rotational crops, such as corn, cotton, rice, and wheat, that are the significant rota tional crops following soybean due to control measures that are available for volunteer soyb ean when they arise (C arpenter et al., 2002; OECD, 2000). Preplant tillage is the firs t management tool for control of emerging volunteer soybean in the spri ng. If volunteer soybean sh ould emerge after planting,

shallow cultivation will control most of the plants and effectively reduce competition with the crop. Several postemergence herbicides also are available to control volunteer soybean (conventional or glyphosate tolerant soybean) in each of the major rotational crops. Table IX-11 provides control ratings on volunteer glyphosate-tolerant soybean for several herbicides used in the major rotational crops.

To provide control of volunteer soybean in corn, postemergence applications of AAtrex (atrazine), Clarity (dicamba), Distinct (diflufenzopyr + dica mba), Hornet (flumetsulam + clopyralid) and Widematch (clopyralid + fluroxypyr) provide excellent control (Zollinger, 2005). In wheat, Bronate Advanc ed (bromoxynil), Clarity (dicamba) and Widematch postemergence provide excellent control of volunteer soybean (Zollinger, 2005).

Volunteer soybean in cotton is normally not a concern. However, hurricanes or other extreme weather conditions can damage a soybean crop preceding cotton production in the Mid-South states, where th e unharvested soybean seed ca n produce volunteer plants. Preplant applications of paraquat or herb icide mixtures containing paraquat will effectively control volunteer glyphosate-tolerant soyb ean (Montgomery et al., 2002; Murdock et al., 2002). Recent research in North Carolina indicates Envoke (trifloxysulfuron) will provi de excellent postemergence c ontrol of soybean containing traits for glyphosate and su lfonylurea herbicide toleran ce in Roundup Ready cotton (York et al., 2005).

Volunteer soybean in rice is rarely a concern due to the combination of preplant tillage, flooding practices, and herbicides used in pr oducing rice. If vol unteer plants should emerge in rice, the postemergence applic ations of Grasp (penoxsulam), Permit (halosulfuron) and Regiment (bispyribac) typi cally used for weed control in rice will effectively alleviate compet ition from volunteer soybean (Dillon et al., 2006).Monsanto Company 09-SY-201U Page 167 of 471 Table IX-11. Ratings for Control of Vo lunteer Glyphosate-Tolerant Soybean in
Labeled Rotational Crops 1

Product Rate (Product/Acre) Soybean

V2 – V3 Soybean

V4- V6

Corn 2

AAtrex 0.38 qts E P 0.50 qts E F
Clarit y 4 fl oz E E

5 fl oz E E

Distinc t 1 oz E G

2 oz E E

Horne t 1 oz E F

2 oz E F-G
Widematch 0.25 pt E G

Wheat 2

Bronate Advance d 0.8 pt E E Clarity 4 fl oz E E

5 fl oz E E

Widematch 0.25 pt E G

Cotton 3

Envoke 0.1 oz E E

Rice 4

Grasp 2 oz E NA

Permit 1 oz E NA Regiment 0.4 oz E E

NA denotes “not applicable.”

1 Weed control ratings: E = Excellent (90 to 99% control), G = Good (80 to 90% control), F =

Fair (65 to 80 control), and P = Poor (40 to 65% control).

2 Zollinger, 2005.

3 York et al., 2005.

4 Dillon et al., 2006.Monsanto Company 09-SY-201U Page 168 of 471 IX.I. Stewardship of MON 87705

Monsanto Company is firmly committed to it s legal, ethical, a nd moral obligation to ensure that its products and technologies ar e safe and environmentally responsible. Monsanto demonstrates this commitment by implementing product stewardship processes throughout the lifecycle of a product and by pa rticipation in the Excellence Through Stewardship SM Program (http://www.excel lencethroughstewardship.org/ )

. These policies and practices include rigorous field complia nce and quality management systems and verification through auditing.

As with all of our products, Monsanto is committed to th e rigorous product stewardship of MON 87705. In keeping with past prac tice, Monsanto will seek regulatory approval for MON 87705 in all key soybean import count ries with a functioning regulatory system to assure global compliance an d support the flow of intern ational trade. Monsanto

continues to monitor other count ries that are key importers of soybean from the U. S., for the development of formal biotechnology a pproval processes. If new functioning regulatory processes are developed, Mons anto will make appropriate and timely regulatory submissions.

As with other value-added specialty soybean , once appropriate approvals are received,

MON 87705-derived varieties wi ll be grown using an appropriate IDP system based on established practices as described in Sectio n IX.B.3. IDP practices are implemented for value-added specialty soybean to capture th e enhanced value of the product and ensure that the end-user or processor receives the soybean with the identity, fatty acid composition of the oil, and desired quality.

Prior to obtaining all key global regulatory approvals, M onsanto will conduct seed testing, variety development and producti on, and oil manufact uring, testing and commercial activities leading to the commercial introduction of soybean varieties developed with MON 87705. Monsanto will work in a closed loop system under contract with partners to plant, harvest and process M ON 87705 and MON 87705-derived soybean for the purpose of producing lo w saturated fat, high oleic oil.

A closed loop system will utilize appropriate processes for containment, documentation and traceability of seed production, planting, harvest, processing and use of the product. Grain production processes wi ll include mass balance and accounting of all planting, harvested seed, secured storage facilities, labe ling of all soybean, training of personnel,

identification and audit of fiel d production sites and acreage, spatial isolation of fields, equipment clean out procedures and documen tation. Soybean processing will include segregation, control and traceabil ity of handling, processing, packaging, and shipping of products and co-products.

Before implementing a closed loop system, Mo nsanto will dialogue with the appropriate

value chain stakeholders in th e countries of production and of use of the product to gain

feedback on and confirm the robustness an d validity of the closed loop production system. Monsanto will not employ the closed loop system without adequate assurance that the system will be effective in contai ning, preventing the escape of, and controllingMonsanto Company 09-SY-201U Page 169 of 471 the disposition of MON 87705 and MON 87705-der ived soybean so it is not comingled with commodity soybean. As part of this process, Monsanto will continue to provide regular updates on MON 87705 to key members of the soybean industry grain trade, processing industry and food industry throughout the regulatory and product development

process.

IX.J. Impact of the Introduction of MON 87705 on Agricu ltural Practices

Introduction of MON 87705 is expected to ha ve no impact on current cultivation and management practices for soybean. The R oundup Ready soybean system, i.e., planting Roundup Ready soybean and applying glyphosate in crop, has become the standard weed control program in U.S. soybean producti on. Currently Roundup Ready soybean is planted on 92% of U.S. soybean acres (USDA-NASS, 2008). Therefore from an agricultural perspe ctive, MON 87705 is similar to the commercial Roundup Ready soybean products used in the U.S. since 1996. For an overview of the cumulative impacts on agricultural practi ces (weed control, tillage and crop rota tion) from deregulation of glyphosate-tolera nt crops see Appendix N.

Changes in the MON 87705 soybean oil profile ar e not anticipated to impact agricultural practices. MON 87705 is a nutritionally impr oved soybean product that is expected to bring added value to consumers that will result in the soybean being sold at a premium. The added value of MON 87705 as a specialty soybean is expected to bring higher returns to growers compared to commodity soybean. As such, MON 87705 will be harvested, processed and marketed using we ll-established IDP met hods to maintain its integrity, purity and value.Monsanto Company 09-SY-201U Page 170 of 471 X. ENVIRONMENTAL CONSEQUENC ES AND IMPACT ON AGRONOMIC

PRACTICES

X.A. Introduction

This section provides a brie f review and assessment of the plant pest potential of MON 87705 and its impact on agronomic pr actices. USDA-APHIS has responsibility, under the Plant Protection Act (7 U.S.C. § 7701-7772), to prevent the introduction and dissemination of plant pests in to the U.S. APHIS regulatio n 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate su bmitted data to determine that a particular regulated article does not present a plant pest risk and no longer should be regulated. If APHIS determines that the regulated article doe s not present a plant pe st risk, the petition can be granted, thereby allowing unre stricted introduction of the article.

The definition of “plant pest” in the Plant Protection Act (PPA) in cludes living organisms that could directly or indirectly injure, da mage, or cause disease in any plant or plant product (7 U.S.C. § 7702[14]). Information in this petition related to plant pest risk

characteristics includes the mechanism of action and changes to plant metabolism and composition, expression and characteristics of the gene products (C P4 EPSPS protein and

the FATB1-A and FAD2-1A RNA), weediness of the regulated article, impacts to NTOs, disease and pest susceptibilities, impact s on agronomic practices, any impacts on the weediness of any other plant with which it can interbreed, and the transfer of genetic information to organisms with which it cannot interbreed.

The regulatory endpoint under th e PPA for biotechnology-deriv ed crop products is not zero risk, but rather a determination that dere gulation of the regulated article is not likely to pose a plant pest risk. The plant pe st risk assessment of MON 87705 is based primarily on eight lines of evidence: (1) insertion of a single functional copy of the inserted expression cassette, (2) characteri zation of the CP4 EPSPS protein expressed in MON 87705 and the improved fatty acid profile, (3) safety of the CP4 EPSPS protein and the improved fatty acid profile, (4) compos itional equivalence of harvested MON 87705 soybean seed as compared to conventi onal soybean, (5) phenotypic and agronomic characteristics demonstrating no increased plant pest potential, (6) negligible risk to NTOs and threatened or endangered species , (7) modern soybean has inherently low plant pest potential, and (8) no greater likelihood to impact agronomic practices, including land use, cultivati on practices, or the management of weeds, diseases, and insects than conventional soybean.

Phenotypic and agronomic char acteristics of MON 87705 were analyzed to determine if it will become any more weedy or invasive re lative to soybean varieties currently on the market approved for unconfined release. A dditional evaluations in cluded the propensity of MON 87705 to become a greater reservoir of plant pests (insects or pathogens) when compared to conventional plants. An asse ssment for potentially adverse impacts of MON 87705 on NTOs, including sy mbiotic soil microorganisms, also was performed. The potential for, and consequences of, gene flow and introgress ion of the genetic construct into sexually compatibile plants or wild relatives, were also evaluated to determine the potential for increased weedy or invasive characteristics in sexually compatible plant species.Monsanto Company 09-SY-201U Page 171 of 471 Molecular characterization of MON 87705 by Southe rn blot analyses demonstrated that a single copy of the T-DNA I and T- DNA II sequences from the transformation vector PV-GMPQ/HT4404 was integrated into the soyb ean genome at a single locus. There were no additional genetic elements, including backbone sequences, from the transformation vector PV-GMPQ/HT4404 detected , linked or unlinked to the intact DNA insert, in MON 87705. The assessment of we ediness potential and gene flow indicated that MON 87705 is no more likely to become a weed than conventional soybean, and MON 87705 is expected to be similar to conve ntional soybean regarding the potential for and impact from gene flow. Due to lack of sexually compatible relatives in the U.S., pollen-mediated gene flow is expected to occur only within cultiv ated soybean. Given the reproductive biology of soybean, pollen-me diated gene flow is expected to be negligible within cultivated soybean. The probability for horizontal gene flow is exceedingly small. Even if it were to occu r, the consequences would be negligible because the CP4 EPSPS protein which confer s herbicide tolerance has no meaningful toxicity to humans and NTOs under the cond itions of use. Transfer of the tandem

FATB1-A and FAD2-1A gene segments is equall y unlikely. As with the cp4 epsps gene, even if transfer were to occur, the result would be negligible because the FATB1-A and

FAD2-1A gene segments do not encode for a protein, nucleic ac ids are generally recognized as safe (GRAS), and th e resulting change in fatty acids results in an oil profile that is similar in composition to other plant-derived oils. An assessment of current agronomic practices in the U.S. indicates that the introduction of MON 87705 will not impact current U.S. soybean cultivation practic es or the management of weeds, diseases, or insects (Section IX).

Using the assessment above, the data and analys is presented in this petition leads to a conclusion that MON 87705 is unlikely to be a plant pest and, therefore, no longer should be subject to regulati on under 7 CFR §340.

APHIS recently has proposed amendments to 7 CFR § 340 that incl ude its noxious weed authority. Because the data show that MON 87705 has no potential to cause injury or damage to protected interests under the noxious weed authority, MON 87705 also would

not be considered a “noxious weed” as defined by the Plant Protection Act.

X.B. Plant Pest Assessment of the MON 87705 Insert and Expressed Substances

X.B.1. Characteristics of the Genetic Insert and the Expressed Protein

Genetic Insert

MON 87705 was produced by Agrobacterium -mediated transformation of soybean with PV-GMPQ/HT4404, which is a binary vector containing 2T-DNAs (Table IV-1). TDNA I contains a cp4 epsps expression cassette and a pa rtial suppression cassette containing the sense segments of the FAD2-1A intron and FATB1-A 5' UTR. T-DNA II contains a partial suppression cassette that consists of the antisense segments of FAD2-1A

and FATB1-A . During plant transformation, the tw o T-DNAs co-integrated at one locus in the soybean genome, creating a singl e DNA insert that contains the single cp4 epsps cassette and a single FAD2-1A and FATB1-A suppression cassette. The chimeric promoter P-FMV/TSF1 consists of enhancer sequences from the 35S promoter of theMonsanto Company 09-SY-201U Page 172 of 471 figwort mosaic virus (FMV) and the promot er from the Tsf1 gene of the plant Arabidopsis thaliana encoding elongation factor EF-1 alpha. The FMV promoter has a history of safe use in transgenic plants (USDA-APHIS, 2006b) and is highly unlikely to promote plant disease. The inserted T- DNA in MON 87705 contai ns left and right border sequences from Agrobacterium tumefaciens , a plant pest. These sequences are well characterized, are noncoding regions and will not cause MON 87705 to promote plant disease (Section IV).

Molecular analyses demonstrate that MON 8770 5 contains one copy of the insert at a single integration locus. No additional elements from th e transformation vector were detected in the genome of MON 87705, incl uding backbone sequence from plasmid PVGMPQ/HT4404. Additionally, the data confirm the organi zation and sequence of the insert, demonstrate the stability of the insert over several generations, and demonstrate that the genomic DNA sequences flanking the 5' a nd 3' ends of the insert are native to the soybean genome. On the basis of these da ta, it is concluded that the expected CP4 EPSPS protein and dsRNA are produced from the inserted DNA.

Mechanism of Action

Monsanto Company has deve loped biotechnology- derived soybean with an improved soybean oil profile. Compared to conventional soybean oil, MON 87705 soybean oil fatty acid (FA) levels are lower for saturate d fats (6% vs. 15% FA), and higher for oleic acid (76% vs. 23% FA) (USDA-NND database at http://www.nad.usda.gov). The increase in monounsaturated fatty acid (ole ic) is accompanied by an overall decrease in polyunsaturated fatty acids (17% vs 60% FA). As descri bed earlier in Section I.C., the improved fatty acid profile in MON 87705 soyb ean oil is achieved through the use of endogenous soybean ( Glycine max L.) gene segments configured to suppress FATB and FAD2 gene expression. MON 87705 contains FATB1-A and FAD2-1A gene segments under the control of a seed promoter, limiting oi l composition modification to this tissue. The assembled gene transcript has an inverted repeat th at produces double stranded RNA (dsRNA) that, via the RNA interference (R NAi) pathway, suppresses endogenous FATB and FAD2 gene expression, thereby producing the desired fatty acid phenotype. Acylacyl carrier protein (ACP) th ioesterases (referred to herein as FATB enzymes) are localized in plastids and hydrol yze saturated fatty acids from the ACP-fatty acid moiety. The suppression of FATB results in a decrease in the transport of the saturated fats out of the plastid, thus retaining their availability for desaturation to 18: 1 oleic acid (see Figure E-1). Therefore, suppression of FATB decreases saturated fat co ntent in the oil as well as increasing oleic acid. Subsequently, this increased amount of oleic acid is either delivered to the oil body or endoplasmic retic ulum for further desa turation. Delta-12 desaturases (referred to as FAD2 enzymes) desaturate 18:1 oleic acid to 18:2 linoleic acid. The suppression of FAD2 in soybean se ed causes reduced desaturation of oleic to linoleic acid thus contributing further to the increase in ol eic while reducing linoleic acid content in the oil. Therefore, the overall result of the suppr ession of these two enzymes is a reduction in saturated 16:0 palmitic and 18:0 stearic fatty acids, an increase in monounsaturated 18:1 oleic acid, and lower le vels of polyunsaturated 18:2 linoleic acid relative to commodity soybean.Monsanto Company 09-SY-201U Page 173 of 471 The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is mediated by dsRNA molecules. Doubl e stranded RNAs are commonly found in eukaryotes, including plants , for endogenous gene suppression and are composed of nucleic acids. Nucleic acids have a long hist ory of safe consumption and are considered GRAS by the U.S. Food and Drug Administra tion. There is no evidence to suggest dietary consumption of RNA is associated wi th toxicity or allergenicity. Moreover, analysis of the DNA segments encoding this ds RNA showed that they lack the sequences required for translation initiati on and protein synthesis. Th e production of a protein from the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several biotechnology-derived plant pr oducts previously deregulated by APHIS were developed using RNA-based suppression mechanisms, incl uding virus-resistant papaya and squash, high oleic soybean, FLAVR SAVR tomatoes, a nd plum trees resistan t to Plum pox virus (FDA, 1994; USDA-APHIS 1994; USDA-APHIS, 1997; USDA-APHIS, 2006; USDAAPHIS, 2007). Based on this information, it is concluded that the inserted DNA and resulting dsRNA are safe and unl ikely to produce a protein or polypeptide. As a result, the RNA-based suppression technology used in MON 87705 poses no novel risks from a food, feed or environmental perspective.

MON 87705 incorporates the cp4 epsps coding sequence derived from the common soil

bacterium Agrobacterium sp. strain CP4. The cp4 epsps coding sequence directs the production of the 5-enolpyruvylshikimate-3- phosphate synthase (termed CP4 EPSPS protein) that is less sens itive to inhibition by glyphosat e compared to endogenous EPSPS in plants. The CP4 EPSPS protein renders Roundup Rea dy soybean tolerant to glyphosate, the active ingred ient in Roundup agricultural herbicides. The CP4 EPSPS protein has been assessed previously as a component in multiple Roundup Ready crops

and has not been found to confer a selectiv e advantage which woul d lead to increased plant pest risk.

Expressed Protein Safety

The CP4 EPSPS protein produced in MON 8 7705 is equivalent to the CP4 EPSPS proteins consumed in food and feed deri ved from other Roundup Ready crops, such as Roundup Ready soybean, that have a history of safe use. It is structurally homologous to EPSPS proteins that are part of the amino acid synthesis pathway of all plants (Devine et al., 1993). The safety of CP4 EPSPS proteins present in bi otechnology-derived crops has been extensively evaluated by Harrison et al. (1996). EPA also has reviewed the safety of the CP4 EPSPS protein and has established a tolerance exemption for the protein and the genetic material necessary for its productio n in or on all raw agricultural commodities (40 CFR § 174.523). This exemption was ba sed on a safety assessment that included rapid digestion in simulated gastric fluids , lack of homology to known toxins and allergens, and lack of toxicity in an acute oral mouse gavage study. A history of safe use is supported by the lack of any documente d reports of adverse effects since the introduction of other Roundup Ready crops in 1 996. Therefore, it is concluded that the CP4 EPSPS protein poses no risk to human or animal health.Monsanto Company 09-SY-201U Page 174 of 471 Protein Expression Levels

CP4 EPSPS mean protein expression levels in MON 87705 samples range from 77 to 530 μg/g dry weight for root, forage, harvested seed and overseason leaf (see Section VI). These levels are comparable to other co mmercialized CP4 E PSPS protein-containing soybean products and confer tolerance to glyphosate found in the agricultural herbicide Roundup. The inverted repeat encoded by the insert in MON 87705 does not code for any proteins. Thus, no new proteins are produced in s oybean containing the MON 87705

trait.

X.B.2. Compositional Characteristics

MON 87705 soybean seed was expected to be compositionally equivalent to conventional soybean except for the intended changes in their fatty acid composition brought about by the suppression of FATB and FAD2 RNAs. Compositional analyses were conducted on seed and forage from MON 87705 samples collected at multiple sites. Additional non biotechnology-deri ved conventional soybean vari eties also were included in the analysis to establish a range of natural variability for each analyte.

The compositional analyses confirmed MON 87705 seed had the intended change in fatty acid composition; that is, decreased levels of the saturated fatty acids, elevated levels of 18:1 oleic acid, and a corresponding decrease in 18:2 linoleic acid. Other components analyzed in MON 87705 seed and forage were compositionally equiva lent to that of conventional soybean control (Section VII). Co llectively, the data established that, with the exception of intended changes in fatty acid levels, MON 87705 is compositionally equivalent to conventional soybean.

Similarly, fatty acid analysis of MON 87705 refined, bleached and deodorized oil

confirmed the intended differen ces for saturated fatty acids, oleic acid, and linoleic acid.

Food and feed derived from M ON 87705 soybean oil will be lower in saturated fats than food and feed derived from commodity soybea n oil, and is considered nutritionally improved. Other components analyzed in MON 87705 processed fractions were compositionally equivalent to that of convent ional soybean control. Collectively, the data established that, with the exception of intended changes in fatty acid levels, MON 87705 is compositionally equiva lent to conventional soybean.

X.B.3. Phenotypic and Agronomic and Envi ronmental Interaction Characteristics

An extensive and robust set of information and data were used to assess whether the introduction of the improved oil profile trait altered the plant pest potential (OECD, 1993) of MON 87705 compared to the conventiona l soybean control, which had a genetic background similar to MON 87705 but did not possess the improved fatty acid profile trait. Phenotypic and agronomic charac teristics of MON 87705 were evaluated and compared to those of the conventional soybean control. These assessments included seed dormancy and germination parameters, plant growth and developm ent characteristics, plant responses to abiotic stressors, polle n morphology and viability and plant symbiont interactions (Section VIII). Results fr om the phenotypic and agronomic assessments demonstrate that MON 87705 does not possess char acteristics that would confer a plant pest risk compared to conventional soybean. Data on e nvironmental interactions alsoMonsanto Company 09-SY-201U Page 175 of 471 indicate that MON 87705 does not confer any biologically meaningful increased susceptibility or tolerance to specific disease, insect, or abiotic stressors, or changes in agronomic and phenotypic characteristics. Taken together, these data support the conclusion that MON 87705 is not likely to pose increased plant pest risk compared to conventional soybean.

Seed Dormancy and Germination

Seed dormancy and germination characteri zation indicated that MON 87705 seed had germination characteristics similar to that of the conventional soybean control. In particular, the lack of hard seed, a well-a ccepted characteristic of ten associated with plants that are weeds, suppor ts a conclusion of no increa sed weediness potential of MON 87705 when compared to conventional soybean.

Plant Growth and Development

Evaluations of plant growth and development characteristics in the field are useful for assessing potential weediness characteris tics such as lodging and pod shattering, indicated there were no significant differences between MON 87705 and the conventional control. Of the remaining 12 growth and development characteristics assessed, there were four si gnificant differences (early stand count, final stand count, days to flowering and 100 seed weight). The differences in these parameters were relatively small in magnitude, and the mean values of MON 87705 were within the range

of the reference varieties for these characte ristics. Thus, the differences in these parameters are unlikely to be biologically meaningful in terms of increased weed potential.

Response to Abiotic Stressors

Comparative field observations between MON 87705 and its conventional control and their response to abiotic st ressors, such as drought, heat stress, high winds, nutrient deficiency, etc., found no diffe rences. Therefore, these fa ctors indicate no increased weediness potential.

Pollen Morphology and Viability

Evaluations of pollen morphology and viabili ty from field-grow n plants may be indicative of increased plant pest potential as th ey relate to the potential for gene flow to, and introgression of the biotec hnology-derived trait into othe r soybean varie ties and wild relatives. These evaluations demonstrated no statistically significant differences between

MON 87705 and the conventional control. Taken together, these comparative

assessments indicate that M ON 87705 is not likely to have increased weed or plant pest potential compared to conventional soybean.

Interactions with NTOs

Evaluation of MON 87705 for potential adverse impacts on NTOs is a component of the plant pest risk assessment. The nature of MON 87705 as a product with no pesticidal activity leads to a conc lusion that all exposed organisms ar e considered to be NTOs. In a 2007 U.S. phenotypic and agronomic study, obs ervational data on environmental

interactions were co llected at select sites for M ON 87705 and a conventional soybean control. In addition, multiple commercial conventional soybean varieties were includedMonsanto Company 09-SY-201U Page 176 of 471 in the analysis to establish a range of natu ral variability for each characteristic. The environmental interactions evaluation (Sect ion VIII) included data collected on plantinsect, plant-disease, and plant-environment in teractions. The results of this assessment indicated that the presence of the improved fatty acid profile and herbicide tolerance traits did not alter plant-ins ect interactions, including bene ficial arthropods and insect pests, nor did it alter disease susceptibil ity of MON 87705 compared to conventional soybean. The lack of differences in plant response to disease damage, arthropod damage, and arthropod pest and benefi cial insect abundance demonstr ate that the introduction of the improved fatty acid profile trait is unlikely to be biologically meaningful in terms of increased pest potential.

In the field, soybean forms a complex sy mbiotic relationship with members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae . This symbiosis results in the

formation of root nodules in which the b acteria reduce or fix atmospheric nitrogenproducing ammonia that can be used by the plant. No significant differences were

detected between MON 87705 and the control fo r the parameters measured, indicating no impact on either the symbiotic relationship or the symbiotic nitrogen-fixing bacteria. A lack of altered pest potential of MON 87705, compared to conv entional soybean, is further supported by an assessment demonstrat ing that the symbiosis between nitrogen fixing bacteria and soybean was not altered as a result of the intr oduction of the improved oil profile and herbicide tolerance traits (S ection VIII). Conse quently, there is no increased plant pest potential due to the nut ritionally improved soybean oil profile or

herbicide tolerance.

These results provide eviden ce that when compared to conventional soybean, the

environmental interactions between M ON 87705 and arthropod pe sts, beneficial arthropods, diseases, and nitr ogen-fixing bacteria were not altered. The lack of significant biological differences in plant response to environm ental interactions indicates that it is unlikely the improved fatty acid profile and herbicide-tolerance traits of MON 87705 will be biologically meaningful in te rms of increased plan t pest potential.

X.C. Weediness Potential of MON 87705

The commercial Glycine species in the U.S. ( Glycine max L.) does not exhibit weedy

characteristics and is not effective in invadi ng established ecosystems. Soybean is not listed as a weed in the major weed refe rences (Crockett, 1977; Holm et al., 1979; Muenscher, 1980), nor is it pres ent on the lists of noxious weed species distributed by the federal government (7 CFR § 360). Soybean does not possess any of the attributes commonly associated with weeds (Baker, 1965), su ch as long persistence of seed in the soil, the ability to disperse, invade, and b ecome a dominant species in new or diverse landscapes or the ability to compete well with native vegetation. Due to the lack of dormancy, a trait that has been removed thr ough commercial breedi ng, soybean seed can germinate quickly under adequate temperature and moisture and potentially can grow as volunteer plants. However, volunteer plants likely would be k illed by frost during autumn or winter of the year they were produced. If they did become established, volunteer plants would not compete well wi th the succeeding crop, and could be controlled readily by either mechanical or ch emical means (OECD, 2000). In addition, since wild populations of Glycine species are not known to exis t in the U.S ., the potentialMonsanto Company 09-SY-201U Page 177 of 471 does not exist for MON 87705 to outcross to wild or weedy rela tives and to alter weediness potential.

In comparative studies between MON 87705 and a conventional soybean control, phenotypic, agronomic, and environmental inter action data were evaluated (Section VIII) for changes that would impact the plant pest potential and, in particular, plant weediness potential. Results of these evaluations s how that there is no fundamental difference

between MON 87705 and the conventional control for traits potentially associated with weediness. Furthermore, comparative field observati ons between MON 87705 and its

conventional control and their response to abiotic stressors, such as drought, heat stress, and high winds, indicated no diffe rences and, therefore, no in creased weediness potential. Collectively, these findings support the conclusion that MON 87705 has no increased weed potential compared to conventional soybe an. Data on environmental interactions

also indicate that MON 87705 does not confer any biological ly meaningful increased susceptibility or tolerance to specif ic disease or insect stressors.

X.D. Potential for Pollen mediated Gene Flow

Gene introgression is a process whereby one or more genes successfully integrate into the genome of a recipient plant. Introgression is affected by many factors, including the frequency of the initial pollination event, e nvironmental factors, sexual compatibility of pollen donor and recipient plan ts, pollination biology, fl owering phenology, hybrid stability and fertility, selection, and the abili ty to backcross repeatedly. Because gene introgression is a natural biological process, it does not constitute an environmental risk in and of itself (Sutherland and Poppy, 2005). Gene introgression must be considered in the context of the transgenes inserted in to the biotechnology-derived plant, and the likelihood that the presence of th e transgenes and their subse quent transfer to recipient plants will result in increased plant pest potential. The potentia l for gene introgression

from MON 87705 is discussed below.

X.D.1. Hybridization with Cultivated Soybean Glycine max

Although soybean is a largely self-pollinated species, low levels of natural crosspollination can occur (Caviness, 1966; OECD, 2000; Ray et al., 2003; Yoshimura et al., 2006). In studies with cultivated soybean where conditions have been optimized to ensure close proximity and fl owering synchrony, natural cr oss-pollination generally has been found to be very low. Most outcros sing occurred with su rrounding plants, and cross-pollination frequencies vary depending on growing s eason and genotype. Insect activity does increase the outcrossing rate, but soybean generally is not a preferred plant for pollinators (Erickson, 1975; Erickson, 1984).

Numerous studies on soybean cross-pollinati on have been conducted, and the published results, with and without supplemental pollinat ors, are summarized in Table X-1. Under natural conditions, cross-polli nation among adjacent plants in a row or among plants in adjacent rows ranged from 0 to 6.3%. In experiments where supplemental pollinators (usually bees) were added to the experiment al area, cross-pollination ranged from 0.5 to 7.74% in adjacent plants or adjacent rows. However, cross-pollination does not occur at these levels over long distances. Cross-pollin ation rates decrease to less than 1.5%Monsanto Company 09-SY-201U Page 178 of 471 beyond one meter from the pollen source, and rapidly decrease with greater distances from the source. The following cross-pollinat ion rates at extended distances have been reported: 0.02% at 8.2 m of separation (Cav iness, 1966), 0.05% at 5.4 m (Ray et al., 2003), and 0% at 6.5 m (Abud et al., 2003).

The potential for cross-pollinati on is limited. This is recognized in certified seed regulations for foundation seed in the U.S., which permit an y distance between different soybean cultivars in the field as long as th e distance is adequate to prevent mechanical mixing (USDA-APHIS, 2006a).

The ecological risk associated with gene flow and introgre ssion from MON 87705 derives from the presence of the cp4 epsps gene and the FAD2-1A and FATB1-A gene segments. The consequence of introgression of the cp4 epsps gene and the FAD2-1A and

FATB1-A gene segments from MON 87705 into other soybean is negligible since, as data presented in this petition confirm, they c onfer no increased plant pest potential to soybean.

X.D.2. Hybridization with Wild Annual Species within Subgenus Soja

The subgenus Soja includes the cultivated soybean Glycine. max and the wild annual species Glycine soja

. Glycine soja is found in China, Taiwan, Japan, Korea, and Russia and can hybridize naturally w ith the cultivated soybean, G. max (Hymowitz, 2004). Hybridization between female G. soja and male G. max was less successful than hybridization in the opposing direction (Dorokhov et al., 2004), where frequency of spontaneous cross pollination in reciprocal combinations of G. max and G. soja varied from 0.73 ( ♀ G. soja × ♂ G. max ) to 12.8% ( ♀ G. max × ♂ G. soja ). Species relationships in the subgenus So ja indicated that F1 hybrids of G. max and G. soja carry similar genomes and are fertile (Singh and Hymowitz, 1989). Abe et al. (1999) note that “natural hybrids between G. max and G. soja are rare ... and hybrid swarms involving both species have never been re ported.” This is also supported by work from Kuroda et al (2008) in which molecular marker s was used and no gene flow from G. max to G.soja was detected. Many barriers exist to natu ral hybridizatio n between soybean and wild relatives, including the highly se lfing nature of both plants, required proximity of wild soybean to cultivated soybean, synchrony of fl owering, and presence of pollinators. As such, it is highly unlikely that naturally occurring, pollen-mediated gene flow and transgene introgression into wild soybean relatives from incidentally released biotechnology-derived soybean will occur at any meaningful frequency.

As described earlier, the s ubgenus Soja also contains an unofficial species, G. gracilis (Hymowitz, 2004). G. gracilis is known only from Northeast China, and is considered to be a weedy or semi-wild form of G. max , with some phenotypic characteristics

intermediate to those of G. max and G. soja

. G. gracilis may be a hybrid between G. soja

and G. max (Hymowitz, 1970). Interspecific fertile hybrids formed by intentional crosses

between G. max and G. soja and between G. max and G. gracilis have been easily obtained (Dorokhov et al., 2004). Although hybridization between G. max and members of the subgenus G. soja can take place, G. soja is not found in North or South America, and it is highly unlikely that gene transfer will occur.Monsanto Company 09-SY-201U Page 179 of 471 X.D.3. Hybridization with the Wild Perennial Species of Subgenus Glycine

Wild perennial species of the Glycine subgenus occur in Austra lia; West, Central and South Pacific Islands; China; Papua, New Guin ea; Philippines; and Taiwan. Therefore, the only opportunities for inter-subgeneric hybridization would occur in areas where those species are endemic (Hymowitz et al., 1992; Hymowitz and Singh, 1992). Nonetheless, the likelihood of in terspecific hybridization between G. max and the wild perennial Glycine species is extremely low because th ey are genomically dissimilar (see Table II-2) and pod abortion is common. From time to time, immature seeds of the crosses have been germinated aseptically in vitro , but the resulting F1 hybrids are slowgrowing, morphologically weak, and completely sterile. Their steril ity is due to poor chromosome pairing. Furthermore, specie s distantly related usually produce nonviable F1 seeds that either have premature death of the germinating seedlings or suffer from seedling and vegetative lethality (Kollipara et al., 1993; Singh and Hymowitz, 1989). In North and South America, it is not possible for gene transfer to occur between cultivated soybean and wild perennial species of Glycine subgenera because th ese wild species do not exist in these regions.

X.D.4. Transfer of Genetic Information to Species with which Soybean Cannot Interbreed (Horizontal Gene Flow)

Monsanto is not aware of any reports regarding the unaided transfer of genetic material from soybean species to othe r sexually incompatible plan t species. The likelihood for horizontal gene flow to occur is exceedingly small. The ec ological risk associated with gene flow and introgress ion from MON 87705 derives fr om the presence of the cp4 epsps gene and the FAD2-1A and FATB1-A gene segments. The cons equence of in trogression

of the cp4 epsps gene and the FAD2-1A and FATB1-A gene segments from MON 87705 into other soybean is negligible since, as da ta presented in this petition confirm, they confer no increased plant pe st potential to soybean.

X.E. Summary of Plant Pest Assessments

Plant pests are defined in th e Plant Protection Act as cert ain living organisms that can directly or indirectly injure , cause damage to, or cause di sease to any plant or plant product (7 U.S.C. § 7702[14]). Characterization data presen ted in Sections III through IX of this petition confirms MON 87705 is not meaningfully different from conventional soybean in terms of pest po tential in its phenotypic, agronomic and environmental interaction characteristics, with the exception of the nut ritionally improved fatty acid profile and glyphosate tolerance. Thus, M ON 87705 is similar to conventional soybean in its plant pest potential. Monsanto is not aware of a ny study results or observations

associated with MON 87705 that would suggest an increased pl ant pest risk would result

from its introduction.

The plant pest assessment was based on multiple lines of evidence developed from a detailed characterization of MON 87705 comp ared to conventional soybean, followed by

a risk assessment on detected differences. Th e risk assessment consid ered various factors including: (1) insertion of a single functional copy of the in serted expression cassette, (2) characterization of the CP4 EPSPS protei n expressed in MON 87705 and the improvedMonsanto Company 09-SY-201U Page 180 of 471 sfatty acid profile, (3) sa fety of the CP4 EPSPS protei n and the improved fatty acid profile, (4) compositional equivalence of harvested MON 87705 soybean seed as compared to conventional soybean, (5) phenotypic and agronomic characteristics demonstrating no increased plant pest poten tial, (6) negligible risk to NTOs and threatened or endangered spec ies, (7) modern soybean has inherently low plant pest potential, and (8) no greater likelihood to imp act agronomic practices, including land use, cultivation practices, or the management of weeds, diseases, and insects than conventional soybean.

Based on the data and information presented in this petition, it is concluded that, like currently deregulated soybean, MON 87705 is highly unlikely to be a plant pest. Therefore, Monsanto Company requests a determination from APHIS that MON 87705 and any progeny derived from crosses be tween MON 87705 and other commercial soybean be granted nonregulated status under 7 CFR § 340.

Table X-1. Summary of Published Litera ture on Soybean Cross Pollination

Distance

from Pollen

Source % CrossPollination Comments Reference

0.3 m 0.04% (estimated per pod) Interspaced plants within a row. Experiment conducted in a single year. Single male and female parental varieties. Percent outcrossing calculated per pod rather than per seed. Woodworth,

1922

0.8 m 0.07 to 0.18% Adjacent rows. Experiment conducted over two years. Several male and female parental

varieties. Garber and Odland, 1926

0.1 m 0.38 to 2.43% Adjacent plants within a row. Experiment conducted in a single year. Several male and female parental varieties. Cutler, 1934

0.1 m 0.2 to 1% Adjacent plants within a row. Experiment conducted in single year at two locations. Several male and female parental varieties. Weber and
Hanson, 1961

0.9 m

2.7 – 4.6 m

6.4 – 8.2 m

10 – 15.5 m 0.03 to 0.44 %

0.007 to 0.04%

0 to 0.02%

0 to 0.01% Frequency by distance was investigated. Experiment conducted over three years. Single male and female parental varieties. Caviness, 1966

0.8 m 0.3 to 3.62% Various arrangements within and among adjacent rows. Experiment conducted over three years. Several male and female parental varieties. Beard and Knowles, 1971

One row (undefined) 1.15 to 7.74% Bee pollination of single-row, small-plots of pollen receptor surrounded by large fields (several acres) of pollen donor soybean. Soybean is not a preferred flower for honeybee. Abrams et al.,

1978Monsanto Company 09-SY-201U Page 181 of 471 Table X-1 (cont.) Summary of Published Literature on Soybean Cross Pollination

Distance

from Pollen

Source % CrossPollination Comments Reference

0.1 – 0.6 m 0.5 to 1.03% (depending on planting design) Bee pollination of soybean grown in various spatial arrangements. Experiment conducted over four years. Several soybean cultivars. Chiang and Kiang, 1987

1.0 m 0.09 to 1.63% Adjacent rows. Experiment conducted over two years. Several male and female parental

varieties. Ahrent and Caviness, 1994

0.5 m

1.0 m

6.5 m 0.44 to 0.45%

0.04 to 1.4%

none detected Frequency by distance was investigated. Experiment conducted in a single year. Single male and female parental varieties. Abud et al.,

2003

0.9 m

5.4 m 0.29 to 0.41%

0.03 to 0.05% Frequency by distance was investigated. Experiment conducted in a single year. Single male and female parental varieties. Ray et al., 2003

0.15 m 0.65 to 6.32%

1.8% Interspaced plants within a row. Experiment conducted in a single year. Single male and female parental varieties. Ray et al., 2003

0.7 m

1.4 m

2.1 m

2.8 m

3.5 m

7.0 m

10.5 m 0 to 0.19%

0 to 0.04%

0 to 0.05%

0 to 0.08%

0 to 0.04%

0 to 0.04%

0 Interspaced plants within a row arranged in small plots. Experiment conducted in a four year period. Single male and two female parental varieties. Yoshimura, et al., 2006Monsanto Company 09-SY-201U Page 182 of 471 XI. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto knows of no study results or obs ervations associated with MON 87705, the improved fatty acid profile or the CP4 EPSPS protein, indicating that there would be an adverse environmental consequence from the introduction of M ON 87705. MON 87705 soybean oil contains a reduced level of satu rated fats, an increase in oleic acid and an

associated decrease in linoleic acid leve ls. The decrease in saturated fats and polyunsaturated fatty acids in MON 87705 soybean oil provi des important options for food companies to develop foods with lower saturated fat and greater food functionality. In addition, these attributes provide key enhancements for biodiesel and industrial applications. MON 87705 also contains the CP4 EPSPS protein that renders the soybean plant tolerant to glypho sate, the active ingredient in the Roundup family of agricultural herbicides. As demonstrated by field results and laboratory tests, the only phenotypic

difference between MON 87705 and conventional soybean is the improved soybean fatty acid profile and gl yphosate tolerance.

The data and information presented in th is petition demonstr ate that MON 87705 is unlikely to pose an increased pl ant pest potential or to ha ve an adverse environmental consequence compared to conventional soyb ean. This conclusion is reached based on multiple lines of evidence developed from a detailed characterization of the product compared to conventional soybean, followed by ri sk assessment on detected differences.

The characterization evalua tions included molecular an d protein analyses, which

confirmed the insertion of a single functional copy of the cp4 epsps and partial suppression cassette at a single locus within th e soybean genome; that the suppression of

FATB and FAD2 RNAs result in the improved soybean fatty acid profile, and that the CP4 EPSPS protein was e xpressed in tissues at levels th at resulted in tolerance to the herbicide glyphosate. Extensive characteri zation of the plant phenotype, including compositional analysis of key nutrients and antinutrients also i ndicated MON 87705, with the exception of the intended fatty acid changes, was unchanged compared to conventional soybean. A history of safe us e of the CP4 EPSPS prot ein is supported by the lack of any documented reports of adve rse effects since the introduction of other Roundup Ready crops in 1996. The EPA previous ly reviewed and established a tolerance exemption for the CP4 EPSPS protein and th e genetic material necessary for its production in or on all raw agricultural commodities.

An endangered species risk assessment concluded MON 87705 is unlikely to have adverse effects on these organisms. Therefor e, the risks for humans, animals, and other NTOs from MON 87705 are negligible under th e conditions of use. Additionally the

introduction of MON 87705 will not adversel y impact cultivation practices or the management of weeds, diseases, and in sects in soybean production systems.

Successful adoption of MON 87705 will provide growers with an opportunity to produce this value-added specialty soybean that produ ces soybean oil with improved stability and a healthier profile to help meet n eeds in food and i ndustrial markets.Monsanto Company 09-SY-201U Page 183 of 471 REFERENCES

Abe J., A. Hasegawa, H. Fukushi , T. Mi kami, M. Ohara, and Y. Shimamoto. 1999. Introgression between and cultivated soybeans of Japan revealed by RFLP analysis for chloroplast DNAs. Economic Botany 53:285-291.

Abrams, R.I., C.R. Edwards, and T. Harri s. 1978. Yields and cr oss-pollination of soybeans as affected by honey bees and alfa lfa leafcutting bees. American Bee Journal

118:555-560.

Abud, S., P.I. Mello de Souza, C.T. Moreir a, S.R.M. Andrade, A.V. Ulbrich, G.R. Vianna, E.L. Rech, and F.J. Lima Aragao. 2003. Pollen dispersal in transgenic soybean plants in the Verrado region. Pesquisa Agropecuaria Brasileira 38:1229-1235.

Adang, M.J., M.J. Staver, T.A. Rocheleau, J. Leighton, R.F. Barker, and D.V. Thompson. 1985. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 a nd their toxicity to Manduca sexta. Gene

36:289-300.

Ahrent, D.K. and C.E. Caviness. 1994. Na tural cross-pollination of twelve soybean

cultivars in Arkansas . Crop Science 34:376-378.

Alexander, M.P. 1980. A vers atile stain for pollen fungi, yeast and bacteria. Stain Technology 55(1): 13-18.

Alibhai, M.F., and W.C. Stallings. 2001. Closing down on glyphosate inhibition - with a new structure for drug discovery. Proceedings of the National Academy of Sciences

U.S.A. 98:2944-2946.

Al-Kaisi, M. 2001. Value of crop rotation in nitrogen management. Iowa State University. http://www.ipm.iastate.edu/ipm /icm/2001/4-23-2001/valuen.html.

Al-Kaisi, M., M.H. Hanna, and M. Tidman . 2003. Crop rotation considerations for 2004 management season rotation. Department of Entomology. Iowa State University, http://www.ent.iastate.edu/ip m/icm/2003/croprotation.html.

Anderson, K.S., and K.A. Johnson. 1990. Kine tic and structural analysis of enzyme intermediates: lessons from EPSP Synt hase. Chemical Reviews. 90:1131-1149.

Anderson, W.P. 1996. Weed Ecology. Page s 27-38 inWeed Science Principles and Applications, Third Edition West Pub lishing Company, St. Paul, Minnesota.

AOSA. 2007. Rules for Testing Seeds. Associa tion of Official Seed Analysts, Lincoln,

Nebraska.Monsanto Company 09-SY-201U Page 184 of 471 AOSCA 2009a. Seed Certification Handbook. A ssociation of Offici al Seed Certifying Agencies. Moline, Illinois.

AOSCA. 2009b. Operational Procedures, Crop Standards and Service Programs.

Association of Official Seed Cert ifying Agencies, Meridian, Idaho.

Appunu, C. and B. Dhar. 2006. Differential symb iotic response of pha ge-typed strains of Bradyrhizobium japonicum with soybean cul tivars. Journal of Mi crobiology 44(3):363-

368.

Aref, S. and D.R. Pike. 1998. Midwest farmer s’ perceptions of crop pest infestations. Agronomy Journal 90:819-825.

ASA. 2009. Soy Stats. American Soybean Association, St. Louis, Missouri.

ASA. 2008. Soy Stats. American Soybean Association, St. Louis, Missouri.

Axelos, M., C. Bardet, T. Libox, A. Le Van Thai, C. Curie, and B. Lescure. 1989. The gene family encoding the Arabidopsis thaliana translation elongati on factor EF-1alpha: molecular cloning, chararacterization and expr ession. Molecular and. General. Genetics.

219:106-112.

Baker, H.G. 1965. Characteristics and modes of origin of weeds. Page 147-172 in The Genetics of Colonizing Species. Baker, H.G. and G.L. Stebbins (eds.). Academic Press,

New York.

Barker, K., I. Chibata, K. Nakayama, K. Takinami, and H. Yamada. 1983. Nucleotide sequence of the T-DNA Region from the Agrobacterium tumefaciens Octopine Ti Plasmid pTi15955. Plant Mo lecular Biology 2:335-350.

Barry, G., M. Taylor, S.R. Padgette, K.H. Kol acz, M. Weldon, D.B. Re, D.Z. Eichholz, K. Fincher, and L. Hallas. 1992. Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants . Biosynthesis and mol ecular regulation of amino acids in plants:139-145.

Barry, G.F., G.M. Kishore, S.R. Padgette, and W.C. Stallings. 1997. Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate s ynthases. U.S.A. Patent 5,633,435.

Baumann, T.T., A.F. Dobbels, W.G. Johnson, M.M. Loux, G.R.M. Nice, and S. J.M. 2008. Weed control guide for Ohio and Indi ana. University Extension Bulletin 789. Purdue University and Ohio State

Beard, B.H., and P.F. Knowles. 1971. Freque ncy of cross-pollination of soybeans after seed irradiation. Crop Science 11:489-492.Monsanto Company 09-SY-201U Page 185 of 471 Boerma, H.R., and J.E. Specht. 2004. Worl d distribution and tr ade of soybean and Managing inputs for peak production; Soybean production in the U.S.A. Pages 3, 502, 507 and 522 in Soybeans: Improvement, Producti on,and Uses. Boerma H.R. and J.E. Specht (eds.). ASA,CSSA-SSSA, Madison, Wisconsin.

Bradford, K.J. 2006. Methods to Maintain Gene tic Purity of Seed Stocks. Agricultural Biotechnology in California Series, Publica tion 8189, University of California.

Bringe, N.A. 2005. Soybean oil composition for biodisease. The Biodiesel Handbook, AOCS Press, Champa ign, Illinois.

Cahoon, E.B. 2003. Genetic enhancement of so ybean oil for industrial uses: prospects and challenges. AgBioForum 6:11-13.

Carpenter, J, A. Felsot, T. Goode, M. Hammig, D. Onstad, and S. Sankula. 2002. Comparative Environmental Impacts of Bi otechnology-derived and Traditional Soybean, Corn, and Cotton Crops. Council for Agricultur al Science and Technology, Ames, Iowa.

CAST 2007. Council for Agricultural Science and Technology. Implications of Gene Flow in the Scale-up and Co mmercial Use of Biotechnology -derived Crops: Economic and Policy Considerations. Issue Paper 37. Ames, Iowa.

Caviness, C.E. 1966. Estimates of natural cross-pollination in Jackson soybeans in Arkansas. Crop Science 6:211-212.

CCC. 2001. An agronomic and economic asse ssment of transgenic canola. Canola Council of Canada. http://www.canola-council. org/gmo_toc.aspx [Accessed July 2, 2009].

CFIA. 1996. The biology of Glycine max (L .) merr. (soybean). Biology Document BIO1996-10, O. Canadian Food Inspection Agency, Ontario

Chardigny J.M., J.L. Sébédio, and O. Berde ux. 1996. Trans Polyunsaturated Fatty Acids: Occurrence and Nutritional Implications. A nnuals of Applied Lipid Reearch 2:1-33.

Chiang, Y.C. and Y.T. Kiang. 1987. Geomet ric position of genotypes, honeybee foraging patterns, and outcrossing in soybean. Botani cal Bulletin of Acad emia Sinica 28:1-11.

Codex. 2003. Guideline For The Conduct Of Fo od Safety Assessment Of Foods Derived

From Recombinant-DNA Plants. Codex Alimentarus. ftp://ftp.fao.org/es/esn/food/guide_pla nts_en.pdf. [Accessed June 8, 2009].

Codex. 2005. Codex Standard for named ve getable oils. Codex Alimentarus.Monsanto Company 09-SY-201U Page 186 of 471 Coruzzi, G., R. Broglie, C. Edwards, and N. Chua. 1984. Tissue-specific and lightregulated expression of a p ea nuclear gene encoding the small subunit of ribulose-1,5- bisphosphate carboxylase. EMBO Journal 3:1671-1679.

Crockett, L. 1977. Wildly Successful Plants : North American Weeds. University of Hawaii Press, Honolulu, Hawaii.

Cutler, G.H. 1934. A simple method for ma king soybean hybrids. Agronomy Journal

26:252-254.

Dalley, C.D., K.A. Renner, and J.J. Kells. 2001. Weed competition in Roundup Ready soybean and corn. Michigan State University, Dept of Crop and Soil Science.

Davis, A., K. Renner, C. Sprague, L. Dyer and D. Mutch. 2005. Integrated Weed Management: “One Year’s Seeding”. Extension Bulletin E-2931. Michigan State University.

Devine. M.D., S.O. Duke and C. Fedtke. 1993. Inhibition of Amino Acid Biosynthesis. Pages 251-294 in Physiology of Herbicide Action. PTR Prentice-Hall, Inc., Englewood Cliffs, New Jersey.

Dillon, T.W., R.C.Scott, N.D. Pearrow, and K.A. Meins. 2006. Eff ect of sulfonylurea rice herbicides on soybeans in 2006 Sout hern Weed Science Society 59.

Dorokhov, D., A. Igantov, E. Deineko, A. Serjapin, A. Ala, and K. Skryabin. 2004. Potential for gene flow from herbicide-resistan t gm soybeans to wild soya in the Russian Far East. Pages 151-161 in Introgression from Genetically Modified Plants into Wild Relatives. Den Nijs, HCM, D. Bartsch, and J. Sweet (eds.). University of Netherlands,

Amsterdam.

Dorrance, A.E., M.A. Draper, and D. Hershm an. 2007. Using foliar fungicides to manage soybean rust. Ohio State University, Columbus, Ohio.

Doyle, J.J., M.A. Schuler, W.D. Godette , V. Zenger, and R.N. Beachy. 1986. The glycosylated seed storage pr oteins of Glycine max and Phaseolus vulgaris. Journal of Biological Chemistry 261:9228-9238.

Dyer, J.M., and R.T. Mullen. 2005. Developmen t and potential of genetically engineered

oilseeds. Seed Science Research 15:255-267.

Eckel, R.H. S. Borra, A.H. Lichtenstein, and S.Y. Yin-Piazza. 2007. Understanding the complexity of trans fatty cid reduction in the American Diet. American Heart

Association Trans Fat Conferen ce. Circulation 115:2231-2246.

Elberhi, A. 2007. The Changing Face of th e U.S. Grain System: Differentiation and Identity Preservation Trends. United Stat es Department of Agriculture - EconomicMonsanto Company 09-SY-201U Page 187 of 471 Research Department, Washington, D.C. (http://www.ers.usda.gov/publications/err35/) [Accessed May 15, 2009].

EPA. 1996. Plant pesticide inert ingredient CP4 enolpyruvylshikimate-3-D and the genetic material necessary for its production in all plants. Federal Register 61:40338-

40340.

Erickson, E.H. 1975. Variability of floral ch aracteristics influen ces honey bee visitation to soybean blossom Crop Science 15:767-771.

Erickson, E.H. 1984. Soybean pollination a nd honey production - a research progress report. American Bee Journal 124:775-779.

FAO/WHO. 2002. Joint FAO/WHO Expert Cons ultation on diet, Nutrition and Chronic

Diseases.

FDA. 1992. Statement of policy: Food derive d from new plant vari eties. Food and Drug

Administration. 57 FR 22984-23005.

FDA. 1994. Biotechnology of f ood. U.S. Food and Drug Ad ministration, Center for Food Safety and Applied Nutr ition, Washington, D.C.

FDA. 2003. Food Labeling: Trans fatty acids in nutritiona l labeling. 68 FR 41434.

FDA. 2006. Food Labeling: Trans fatty acids in nutrition labeling, nutrient content claims,

and health claims. U.S. F ood and Drug Administration, Wa shington, D.C. 68 FR 41434-

41506.

Fillatti, J.J., N.A. Bringe, and K. Dehesh. 2003. Nucleic acid constructs and methods for producing altered seed oil compositions. International Patent WO 2003/080802 A3.

Fling, M.E., J. Kopf, and C. Richards. 1985. Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase.

Nucleic Acids Res earch 13:7095-7106.

Frankel, E.N., 2005. Pages 21, 201-205 in Lipid Oxidation. The Oily Press, Bridgewater, England.

Franz, J., M.K. Mao, and J.A. Sikorski. 1997. Glyphosate: a Unique Global Herbicide. American Chemical Society, Washington, D.C.

Gage, D.J. 2004. Infection and invasion of roots by symbiotic, nitr ogen-fixing Rhizobia during nodulation of temperate legumes. Microbiology and Molecu lar Biology Review

68:280-300.Monsanto Company 09-SY-201U Page 188 of 471 Garber, R.J. and T.E. Odla nd. 1926. Natural crossing in soybeans. Agronomy Journal

18:967 970.

Gianessi, L.P. 2005. Economic and herbicide use impacts of glyphos ate-resistant crops. Pest Management Science 61:241-245.

Giza, P.E., and R.C. Huang. 1989. A se lf-inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene 78:73-84.

Gunstone, F.D. 1994. Fatty acid structure. Pages 1-20 in The Lipid Handbook. 2nd ed. F.D. Gunstone, J.L. Harwood and F.B. Padley (eds.). Chapman & Hall, Cambridge, Great

Btitain.

Gurley. 1979. Sequence organization of th e soybean genome. Biochimica et Biophysica

Acta 561:167-183.

Harrison, L.A., M.R. Bailey, M.W. Naylor, J.E. Ream, B.G. Hamm ond, D.L. Nida, B.L. Burnette, T.E. Nickson, T.A. Mitsky, M.L. Ta ylor, R.L. Fuchs, and S.R. Padgette. 1996. The expressed protein in glyphosate-tole rant soybean, 5-enolpyruvylshikimate-3- phophate synthase from Agrobacter ium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice. Journal of Nutrition 126: 728-740.

Haslam, E. 1993. Shikimic Acid: Metabolism and metabolites. John Wiley and Sons, Chichester, England.

Heatherly, L.G., and H.F. Hodges. 1999. Soybean production in the Midsouth. CRC Press, Boca Raton, Florida.

Helsel, Z.R. and H.C. Minor. 1993. Soybean Production in Missour i. University of Missouri Department of Agronomy. Publication G4410 http://extension.missouri.edu/explore/a gguides/crops/g04410.htm [Accessed January 7, 2009].

Hermann, F.J. 1962. A revision of the genus Glycine and its immedi ate allies. United States Department of Agriculture Technical Bulletin 1268:1-79.

Higley, L.G., and D.J. Boethel. 1994. Handbook of Soybean Insect Pests. The Entomological Society of America, Lanham, Maryland.

Hileman, R.E., A. Silvanovich, R.E. Good man, AE.A. Rice, G. Holleschak, J.D. Aastwood, and S.L. Hefle. 2002. Bioinforma tic methods for allergenicity assessment using a comprehensive allergen database. International Archiv es of Allergy and Immunology 128:280-291.

Ho, T. 1969. The loess and the origin of Chinese agriculture. Am erican Historical

Review 75:1-36.Monsanto Company 09-SY-201U Page 189 of 471 Hoeft, R.G., E.D. Nafziger, R.R. Johnson, and S.R. Aldrich. 2000. Soybean as a crop. Pages 31,36,38,39,41,43,47,86,89,93,96,120,208 in Modern Corn and Soybean.

Production MCSP Publications , Champaign, Illinois.

Holm, L., J.V. Pancho, J.P. Herberger, and D. L. Plucknett. 1979. Intr oduction. Pages i-vii in A Geographical Atlas of World Weeds. John Wiley and Sons, New York.

Hu, F.B., M.J. Stampfer, J.E. Manson, E. Rimm, G.A. Colditz, B.A. Rosner, C.H. Hennekens, and W.C. Willett. 1997. Dietary fat intake and the risk of coronary heart disease in women. The New England Jour nal of Medicine 33 7(21):1491- 1492.

Hymowitz, T. 1970. On domestication of soybean. Economic Botany 24:408-421.

Hymowitz T., 2004, Speciation and cy togenetics. Pages 97-136 in Soybeans: Improvement, Production,and Uses. Boerma H.R. and J.E. Specht (eds.). ASA,CSSA-SSSA, Madison, Wisconsin.

Hymowitz, T., and C.A. Newell. 1981. Taxonom y of the genus Glycine, domestication and uses of soybeans. Economic Botany 35:272-288.

Hymowitz, T., and R.J. Singh. 1987. Taxonom y and Speciation. Soybean Monograph, Soybeans: Improvement, Production and Uses:23-48.

Hymowitz, T., R.J. Singh, and R.P. Larkin. 1990. Long distance dispersal: The case for the allopolyploid Glycine tabacina Benth. and G. tomentella Hayata in the west-central pacific. Micronesia 23:5-13

ILSI. 2004. Nutritional and Safety Assessm ents of Foods and Feeds Nutritionally Improved through Biotechnology. http ://www.ift.org/cms/?pid=1000362.

IOM. 2002. Dietary fats: Total fats and fatty acids. Pages 8-57 and 422-541 in Dietary Reference Intakes for Energy, Carbohydrate, Fibe r, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. Panel on Macronutrients, . Institute of Medicine. National Academies Press, Washington D.C.

ISG. 2006. Soybean data ranked by percentage of no-till. Indiana State Government. http://www.in.gov/isda/files /Soybean_Rank_Percentage.pdf [Accessed July 8, 2009].

ISO. 2009. Selection and Use of the ISO 9000 Family of Standa rds. International Organization for Standardization. http:/ /www.iso.org [Accessed Jan. 9, 2009].

Israel D.W., J.N. Mathis, W.M. Barbour, G. H. Elkan. 1986. Symbiotic effectiveness and

host-strain interactions of Rhizobium fr edii USDA 191 on different soybean cultivars. Applied and Environmental Microbiology 51(5):898-903.Monsanto Company 09-SY-201U Page 190 of 471 John, M.E. and G. Keller. 1995. Characteriza tion of mRNA for a prol inhe-rich protein of cotton fiber. Plant Physiology 108:669-676.

Jordan, N., D.A. Mortensen, D.M. Prenzlow and K.C. Cox. 1995. Si mulation analysis of crop rotation effects on weed seedbanks. American Journal of Botany. 82:390-398.

King, C.A., L.C. Purcel, and E.D. Vories. 2001. Plant growth and nitrogenase activity of glyphosate-tolerant soybeans in response to foliar glyphosate applications. Agronomy

Journal 93:179-186.

Kinney, A.J. 1996. Development of geneti cally engineered soybean oils for food applications. Journal of Food Lipids 3:273-292.

Kinney, A.J. 1998. Plants as industrial chemi cal factories- new o ils from genetically engineered soybeans. Lipid 100.

Kishore, G., D. Shah, S. Padgette, G. Della-C ioppa, C. Gasser, D. Re, C. Hironaka, M. Taylor, J. Wibbenmeyer, D. Eichholtz, M. Hayford, N. Hoffmann, X. Delannay, R. Horsch, H. Klee, S. Rogers, D. Rochester, L. Brundage, P. Sanders, and R.T. Fraley. 1988. 5-enolpyruvylskikimate 3-Phosphate sy nthase. Pages 37-48 in Biotechnology for Crop Protection. Hedin, P.A., J.J. Menn, and R.M. Hollingworth (eds.). American Chemical Society Series No. 379.

Klee, H.J., Y.M. Muskopf, and C.S. Gasser. 1987. Cloning of an Ar abidopsis thaliana gene encoding 5-enolpyruvylshikimate- 3- phosphate synthase: sequence analysis and manipulation to obtain glyphosat e-tolerant plants. Molecu lar Genetics and Genomics

210:437-442.

Knothe, G. 2005. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Pro cessing Technology 86:1059-1070.

Kollipara, K.P., R.J. Singh, and T. Hymow itz. 1993. Genomic diversity in aneudiploid (2n=38) and diploid (2n= 40) Glycine tomentella revealed by cytogenetic and biochemical

methods. Genome 36:391-396.

Kris- Etherton, P. 1995. Trans Acids and Co ronary Heart Disease Risk. Report of the expert panel on trans fatty acids and coronary heart disease, American Journal of Clinical

Nutrition 62:655S-708S.

Kuroda, Y., A. Kaga, N. Tom ooka, and D.A. Vaughan. 2008. Gene flow and genetic structure of wild soybean ( Glycine soja ) in Japan. Crop Sci. 48:1071-1079.

Lackey, J.A. 1981. Phaseoleau DC. Pages 301- 327 in Advances in legume systematics, Part I, R.M. Polhill and R.H. Raven, (eds.). Royal Botanic Gardens, London.Monsanto Company 09-SY-201U Page 191 of 471 Lee, C. and J. Herbek. 2004. Specialty s oybean production and management in Kentucky. University of Kentucky Coopera tive Extension Service.

Lee, J.S., and D.P.S. Verma. 1984. Stru cture and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploidizat ion. EMBO Journal 3:2745-2752.

Lersten, N.R. and J.B. Carlson. 2004. Vege tative morphology. Paes 15-57 in Soybeans: Improvement, Production, and Uses. Boerma, H.R. and J.E. Specht (eds.). Agronomy Monograph 15. ASA, CSSA, and WSSA, Madison, Wisconsin.

Levin, J.G., and D.B. Sprinson. 1964. The enzymatic formation and isolation of 3- enolpyruvylshikimate-5-phosphate. Journa l of Biological Chemistry 239:1142-1150.

Lichtenstein, A.H., L.J. Appel, M. Brands, M. Carnethon, S. Daniels, H.A. Franch, B. Franklin, P. Kris-Etherton, W.S. Harris, B. Ho ward, N. Karanja, M. Lefevre, L. Rudel, F. Sacks, L. Van Horn, M. Winston, and J. Wylie-Rosett. 2006. Diet and lifestyle

recommendations revision 2006: a scientific statement from the American Heart

Association Nutrition Committee. Circulation 114:82-96.

Liener, I. E. 2000. Non-nutritive factors and bioactive compounds in soy. Pages 13–45 in Soy in Animal Nutrition. Drackley,J.K. and I.E. Liener, I. E. (eds.). Animal Science Society, Savoy, IL.

Liener, I.E. 1994. Implications of antinutri tional components in soybean foods. Critical

Reviews in Food Science a nd Nutrition 34(1):31-57.

Liener, I.E. 1955. The photometric determina tion of the hemagglu tinating activity of soyin and crude soybean extracts. Archives of Biochemistry and Biophysics 54:223-231.

Lingenfelter, D.D., and N.L. Hartwig. 2003. Introduction to Weeds and Herbicides. Louisiana State University, Univ ersity Park, Pennsylvania.

Lu, B.-R. 2004. Conserving bi odiversity of soybean gene pool in the biotechnology era. Plant Species Biology 19:115-125.

Martinell, B.J., L.S. Julson, C.A. Emler, Y. Huang, D.E. McCabe, and E.J. Williams. 2002. Soybean agrobacterium transforma tion method. U.S. Patent 6,384,301.

Massey, R.E. 2002. Identity preserved crops . Iowa State University Extension. http://www.extension.iastate.edu/ agdm [Accessed June 1, 2009].

Messina, M. 2001. Isoflavones. United Soybean Board, St. Louis, Missouri.

Metcalfe, D.D., J.D. Astwood, R. Townsend, H.A. Sampson, S. L. Taylor, and R.L. Fuchs. 1996. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Critical Reviews in Food Science and Nutrit ion 376(s):S165-S186.Monsanto Company 09-SY-201U Page 192 of 471 Montgomery, R.F., R.M. Hyayes, C.H. Ti ngle, and J.A. Kendig. 2002. Control of glyphosate-tolerant soybean (Glycine max) in no-till Roundup Rea dy cotton (Gossypium hirsutum L.) in Proceedings of the Beltw ide Cotton Conference National Cotton Council of America, Memphis, Tennessee.

MSA. 2009. Market Trends for IP Crops . Midwest Shippers Association. http://www.mnshippers.com/html/news.cfm?ID=4 [Accessed May 28, 2009].

Muenscher, W.C. 1980. Weeds. Cornell University Press, Ithaca, New York.

Mullin, J.W. abd W. Xu. 2001. Study of s oybean seed coat components and their relationship with water absorption. Jour nal of Agricultural Food Chemistry. 49:5331-

5335.

Murdock, E.C., M.A. Jones, and R.F. Gr aham. 2002. Control of volunteer glyphosate (Roundup)-tolerant cotton and soybean in R oundup Ready cotton. in Proceedings of the 2002 Beltwide Cotton Conferences, Memphis, Tennessee, National Cotton Council.

Muth, M.K., D. Mancini, and C. Viator. 2003. The role of identity -preservation in foodmanufacturer responses to bi oengineered foods. Journal of Food Distribution Research 34(1):43-49.

NDSU. 2002. Soybean production guide for No rth Dakota and Northwestern Minnesota. North Dakota State University Extension Se rvice and North Dakota Soybean Council. http://www.ag.ndsu.edu/pubs/plantsci/rowcr ops/a1172.pdf [Accesse d May 19, 2009].

Nelson, R. 2001. Variation in isoflavones in seeds of domestic and exotic soybean germplasm. ASA-CSSASSSA Annu al Meeting, October 2001, Charlotte, North Carolina.

Nelson, E. and T. Stone. 2003. Petition fo r determination of nonregulated status: Roundup Ready creeping bentgrass (Agrostis stolonifera L.) Ev ent ASR368. The Scotts Company, Gervais, Oregon, and Monsan to Company, St. Louis, Missouri. http://www.aphis.usda.gov/brs/aphis docs/03_10401p.pdf [Accesse d July 7, 2009].

Nice, G., and B. Johnson. 2005. Indiana's T op Ten Most Problematic Weeds. Purdue Extension Weed Science. http://www.bt ny.purdue.edu/WeedScience/2005/topten05.pdf [Accessed May 19, 2009].

OECD. 1993. Safety considerations for Biotechnology: scale-up of crop plants. Organization for Economic Co-opera tion and Development, Paris.

OECD. 1998. Report of the OECD workshop on the toxicological and nutritional testing

of Novel Foods.1-48.Monsanto Company 09-SY-201U Page 193 of 471 OECD. 2000. Consensus document on the biolo gy of Glycine max (L.) merr. (soybean). OECD ENV/JM/MONO(2000)9. Organiza tion for Economic Co-operation and Development, Paris.

OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean: key food and feed nutrients and anti-nutrients. OECD ENV/JM/MONO(2001)15.

Padgette, S.R., D. Re, G. Barry, D. Eichholtz, X. Delannay, R.L. Fuchs, G. Kishore, and R.T. Fraley. 1996a. New weed control opportu nities: Development of soybeans with a Roundup Ready gene. Pages 53-84 in Herbicide- Resistant Crops. Duke, S.O. (ed.). CRC Press, Boca Raton, Florida.

Padgette, S.R., N.B. Taylor, D.L. Nida, M.R. Bailey, J. MacDonald, L.R. Holden, and R.L. Fuchs. 1996b. The composition of glyphosate-tolerant soybean seeds is equivalent to that of conventional soybeans. Journal of Nutrition 126:702-716.

Pedersen, P. 2008a. Row Spacing. Iowa State University Extension. http://extension.agron.iastate.edu/ soybean [Accessed: May 9, 2008].

Pedersen, P. 2008b. Managing soybean cyst nema tode. Iowa State University Extension. http://extension.agron.iastat e.edu/soybean/documents/SCN. pdf [Accessed:May 9, 2008].

Pedersen, P. 2004. Soybean growth and develo pment. Iowa State University Extension.

Pimentel, D. 1991. CRC handbook of pest management in agriculture – 2nd edition.

Volume III. CRC Press. Boca Raton.

Potts, H.C., J. Duangpatra, W.G. Hairston, a nd J.C. Delouche. 1978. Some influences of hardseededness on soybean seed quality. Crop Science 18:221-224.

Prather, T.S., J.M. Ditomasom and J.S. Ho lt. 2000. Herbicide resistance: definition and management strategies. University of Calif ornia, Division of Ag riculture and Natural

Resources.

Pritchett, J.J., J. Fulton, R. Beyers, L. Pederson, and L. Lawson. 2002. Specialty Corn and Soybeans: Production and Marketing in Indiana. EC-714. Purdue University Cooperative Extension Service.

Ray, J.D., T.C. Kilen, A.C. Abel, and R.L. Paris. 2003. Soybean natural cross-pollination

rates under field conditions. Environm ental Biosafety Research 2:133-138.

Richins, R.D., H.B. Scholthof, and R.J. Shepherd. 1987. Sequence of figwort mosaic virus DNA (caulimovirus group). Nucl eic Acids Research 15:8451-8466.Monsanto Company 09-SY-201U Page 194 of 471 Ross, M.A. and C.A. Lembi. 1985. Applied Weed Science. Burgess Publishing Co., Minneapolis.

Salomon, S. and H. Puchta. 1998. Captur e of genomic and T-DNA sequences during double-strand break repair in somatic pl ant cells. EMBO Journal. 17:6086-6095.

Sambrook, J. 1989. Agarose gel electrophoresis. Chapter 5, Protocol 1. in Molecular Cloning, 3rd edition Cold Spring Harbor Laboratory Press, Woodbury, New York.

Schonbrunn, E., S. Eschenburg, W.A. Shuttlewort h, J.V. Schloss, N. Amrhein, J.N.S. Evans, and W. Kabsch. 2001. Interaction of the herbicide glyphosate with its target enzyme EPSP synthase in atomic detail. Proceedings of the National Academy of

Sciences U.S.A. 98:1376-1380.

Scott, O.S., and S.R. Aldrich. 1970. Page s 16, 18, 67, 151, 152 in Modern Soybean production. The Farm Quarte rly, Cincinnati, Ohio.

Silvanovich, A., M.A. Nemeth, P. Song, R. Herman, L.Tagliani, and G.A. Bannon. 2006. The value of short amino acid sequence matches for prediction of pr otein allergenicity. Toxicological Sciences 90:252-258.

Singh, R.J., and T. Hymowitz. 1989. The genom ic relationships between Glycine soja Sieb. and Zucc., G. max (L.) Merr., and G. gracilis Skvortz. Plan t Breeding 103:171-173.

Singh, R.J., H.H. Kim, and T. Hymowitz. 2001. Distribution of rDNA loci in the genus Glycine Willd. Theoretical Applied Genetics 103:212-218.

Skorupska. 1989. Detection of ribosomal RNA genes in soybe an, Glycine max (L.) Merr, by in situ hybridizat ion. Genome 32:1091-1095.

Smyth, S. and P.W.B. Phillips. 2002. Pr oduct differentiation alte rnatives: Identify preservation, segregation, and tra ceability. AgBioForum 5(2):30-42.

Sonka, S., K.I. Bender and D.K. Fisher. 2004. Economics and marketing. Pages 919-948 in Soybeans: Improvement, Production,and Uses. Boerma H.R. and J.E. Specht (eds.). ASA,CSSA-SSSA, Madison, Wisconsin.

Soyatech. 2008. Statistics. Soyatech, Manitoba, Canada.

Stalker, D.M., C.M. Thomas, and D.R. Helinsk. 1981. Nucleotide sequence of the region of the origin of replicatio n of the broad host range plas mid RK2. Molecular Genenetics

and Genomics 181:8-12.

Stallings, W.C., S.S. Abdel-Me guid, L.W. Lim, H.S. Shieh, H.E. Dayringer, N.K. Leimgruber, R.A. Stegeman, K.S. Anderson, J.A. Sikorsi, S.R. Padgette, and G.M.

Kishore. 1991. Structure and topological symmetry of the glyphosate target 5-Monsanto Company 09-SY-201U Page 195 of 471 enolpyruvylkikimate-3-phosphate synthase: a dis tinctive protein fold. Proceedings of the National Academy of Sciences U.S.A. 88:5046-5050.

Steffen, D. 2004. USDA Plant Variety Protection Certificate for Glycine max (L.). Merr.,

No. 200400321 for SN70025.

Steinrücken, H., and N. Amrh ein. 1984. 5-enolpyruvylshikim ate-3-phosphate synthase of Klebsiella pneumoniae. European Journal of Biochemistry. 143:351-357.

Steinrücken, H.C., and N. Amrhein. 1980. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate sy nthase. Biochemical Biophysical Research

Communications 94:1207-1212.

Sundstrom, F.J., J. Williams, A. Van De ynze, and K.J. Bradford. 2002. Identity preservation of agricultural commodities. Division of Agricultural and Natural Resources Publication 8077. University of California-Oakland.

Sutcliffe, J.G. 1979. Complete Nucleotide Sequence of the Escherichia coli Plasmid pBR322. Cold Spring Harb Sy mp Quant Biol. 43:77-90.

Sutherland, J.P. and G.M. Poppy. 2005. Qu antifying exposure. Pages 186-208 in Gene Flow from GM Plants. G.M. Poppy and M.J. Wilkinson (eds.), Blackwell Publishing, Ames, Iowa.

TCM. 2008. Nightshade: Threat to hawr vest and export. Top Crop Manager. http:/www.topcropmanager.com [Accessed July 15, 2009].

Thomas, K., M. Aalbers, G.A. Bannon, M. Bart els, R.J. Dearman, D.J. Esdaile, T.J. Fu, C.M. Glatt, N. hadfield, C. Hatzos, S.L. Hefl e, J.R. Heylings, R.E. Goodman, B. Henry, C. Herouet, M. Holsapple, G.S. Ladies, T.D. Landry, S.C. MacIntosh, E.A. Rice, L.S. Privalle, H.Y. Steiner, R. Teshima, R. Va n Ree, M. Woolhiser, and J. Zawodny. 2004. A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Re gulatory Toxicology and Pharmacology 39:87-

98.

University of Illinois. 2002. Weeds on the horizon. University of Illinois Extension, Champaign, Illinois.

UK. 2009. Specialty soybeans. University of Kentucky College of Agriculture, Cooperative Extension Service.

USDA. 1973. Energy Value of Foods. Pages 2-11 in Agricultural Handbook No. 74. United States Department of Agriculture, Washington, DC.

USDA. 1970. Forage Fiber Analyses. Pages 1-18 in Forage Fiber Analyses, Agricultural Handbook 379. United States Department of Agriculture, Washington, DC.Monsanto Company 09-SY-201U Page 196 of 471 USDA-APHIS. 1997. Availability of Determ ination of Nonregulated Status for Genetically Engineered Soybeans. U.S. Depa rtment of Agriculture, Washington, D.C. 62

FR 27580-27581.

USDA-APHIS, 1994. Availability of Determin ation of Nonregulated Status for Virus Resistant Squash. U.S. Department of Agriculture, Washington, D.C.

USDA-APHIS. 2003. Preliminary Risk Assessm ent on the Petition for a Determination of Nonregulated Status fo r Creeping Bentgrass (Agrostis stolonifera) genetically engineered (Event ASR368) for tolerance to the herbicide glyphosate submitted by Monsanto Company and the Scotts Company. U.S. Department of Agriculture, Washington, D.C. http://www.aphis.usda .gov/brs/aphisdocs/ 03_10401p_ra.pdf [Accessed July 7, 2009].

USDA-APHIS. 2006. Availability of Petit ion and Environmental Assessment for Determination of Nonregulated Status for Pl um Genetically Engineered for Resistance to Plum Pox. U.S. Department of Agricu lture, Washington, D.C. 71 FR 28296-28298.

USDA-APHIS. 2006a. Soybean.U.S. Department of Agriculture, Washington, D.C. http://www.aphis.usda.gov/br/soybean. html [Accessed January 20, 2006].

USDA-APHIS. 2006b. Environmental Assessment: In response to Monsanto petition 06- 178-01p seeking a Determination of Non-re gulated Status for Roundup Ready2 Yield Soybean MON 89788. U.S. Department of Agriculture, Washington, D.C.

USDA-APHIS. 2007. Determination of nonre gulated status for plum genetically engineered for resistance to plum pox vi rus. U.S. Department of Agriculture, Agricultural Research Service; 72 FR 38556-38557. http://www.aphis.usda.gov/brs/fedregis ter/BRS_20070713b.pdf [Accessed June 1, 2008].

USDA-ARS. 2006. Forage soybean cultivars: a s ource of high protein livestock feed. U.S. Department of Agriculture, Washington, D.C

USDA-ERS. 2007. Adoption of gene tically engineered crops in the U.S. U.S. Department of Agriculture, Washington, D.C

USDA-ERS. 2006. Soybean production costs and re turns per planted crop acre, by region, excluding government payments for 2006. U. S. Department of Agriculture-Economic Research Service. (http:// www.ers.usda.gov/data/costsandr eturns/Soy_all.xls) [Accessed June 26, 2009].

USDA-ERS. 2005. USDA soybean baseline 2005-14. http://www.ers.usda.gov/briefing/soybeanso ilcrops/2005baseline.htm [Accessed June 10, 2006].Monsanto Company 09-SY-201U Page 197 of 471 USDA-ERS. 2001. How much U.S. cropland acr eage is under crop residue Management. Briefing Room. U.S. Department of Ag riculture Economic Research Service. (http://www.ers.usda.gov/Briefing/AgChemical s/soilmangement.htm) [Accessed June 24,

2009

USDA-NASS. 2009a. Crop Production 2008 Summary. U.S. Department of Agriculture, Washington, D.C.

USDA-NASS. 2009b. Soybeans : State and Count ry Statistics. U.S. Department of Agriculture, National Agricultural Statistics Service. http://www.nass.usda.gov/QuickStats/inde xbysubject.jsp?Text1 =&site=NASS_MAIN&s elect=Select+a+State&Pass_name=&Pass _group=Crops+%26+Plants&Pass_subgroup=F ield+Crops [Accessed March 20, 2009].

USDA-NASS. 2008a. Acreage. U.S. Depart ment of Agriculture, Washington, D.C.

USDA-NASS. 2007. Acreage 2007 (June report) . U.S. Department of Agriculture National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2006a. Crop production 2005 summar y. U.S. Department of Agriculture National Agricultural Statistics Service.

USHHS.1988. The Surgeon General's Report on Nu trition and Health. U.S. Department of Health and Human Services, Public Health Service, Washington, D.C. http://profiles.nlm.nih.gov/NN/B/C/ Q/G/ [Accessed June 2, 2009].

USHHS. 2005. Dietary Guidelines for Am ericans. HHS Publication No: HHS-ODPHP2005-01-DGA-A. U.S. Department of Health and Human Services, Washington, D.C. www.health.gov/dietaryguideli nes [Accessed June 7, 2009].

Weber, C.R. and W.D. Hanson. 1961. Natura l hybridization with and without ionizing radiation in soybeans. Crop Science 1:389-392.

Webster, T.M., M. Patterson, J. Everest, J. Ferrell, B. Brecke, A.S. Culpepper, E.P. Prostko, J.D. Green, J.R. martin, E. Webster, S. Kelly, J. Griffin, D. Sanders, J. Byrd, A. Kendig, A. York, D. Jordan, L. Fisher, C. Medlin, D. Murray, J. Norsworthy, J. Chapin, L. Nelson, and L. Steckel. 2005. Weed survey : Southern states 2005: Broadleaf crops section (cotton, peanut, soybean, tobacco, a nd forestry). Pages 291-306 in Southern Weed Science Society Proceedings.

WHO. 2003. Diet, Nutrition a nd the Prevention of Chronic Di seases. Report of a joint WHO/FAO Expert consultation. WHO Technical Report Series 915:55-56.

Woodworth, C.M. 1922. The extent of natura l cross-pollination in soybeans. Agronomy

Journal 14:278-283.Monsanto Company 09-SY-201U Page 198 of 471 Wrather, J.A., W.C. Stienstra, and S.R. Koenning. 2000. Soybean disease loss estimates

for the United States from 1996 to 1998. Cana dian Journal of Plant Pathology 23:122-131.

WSSA 1992. Weed Science Society of Ameri ca. Tables. Pp 88, 94, 103, 107, 118, 123, 130, 145, and 393-401. In Crop Losses Due to W eeds in the United States. Bridges, D.C. (ed.). Weed Science Society of America, Champaign, Illinois.

York, A.C., J.B. Beam, and A.S. Culpe pper. 2005. Control of volunteer slyphosateresistant soybeans in cotton. J ournal of Cotton Science 9:102-109.

Yoshimura, Y., K. Matsuo, and K. Yasuda . 2006. Gene flow from GM glyphosatetolerant to conventional soybeans under field conditions in Japan. Environ. Biosafety

Res. 5:169-173.

Zambryski, P., A. Depicker, K. Kruger, and H.M. Goodman. 1982. Tumor induction by Agrobacterium tumefaciens: Analysis of the boundaries of T-DNA. Journal of Molecular and Applied Genetics 1:361-370.

Zhang, L.X., S. Kye-boahen, J. Zhang, and C.E. Watson. 2004. Redefining zones of adaptation of soybean maturity groups in the U.S. 2004 An nual Meeting Abstracts (CDRom). ASA, CSSA, and SSSA, Madison, Wisconsin.

Zollinger, R.K. 2005. North Dakota Weed Control Guide. http://www.ag.ndsu.edu/weeds/w253/ w253w.htm [Accessed June 28, 2006].

Zollinger, R.K. 2000. Survey of Weeds in North Dakota. Extension Weed Specialist, North Dakota State University. http:/ /www.ag.ndsu.nodak.edu/ weeds/ER83/ER83.htm [Accessed June 28, 2006].Monsanto Company 09-SY-201U Page 199 of 471 APPENDICESMonsanto Company 09-SY-201U Page 200 of 471 Appendix A. USDA Notification

Field trials of MON 87705 were conducted in the U.S. since 2005. The protocols for these trials include field performance, breeding and observation, agronomics, and generation of field materials an d data necessary for this petition. In addition to the phenotypic assessment data pr ovided for MON 87705, observati onal data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2007-2008 seasons, are still in prepara tion. A list of tr ials conducted under

USDA notification and the status of the final reports for these trials are provided in

Table A-1.Monsanto Company 09-SY-201U Page 201 of 471 Table A-1. USDA Notifications Approve d for MON 87705 and Status of Trials

Conducted under These Notifications

USDA No. Effective Date Release Site (State) Trial Status

2005

05-220-03n 9/28/2005 PR Submitted to USDA

05-242-05n 9/28/2005 HI Submitted to USDA

2006 06-033-05n 5/18/2006 HI(3) Submitted to USDA
06-045-13n 5/18/2006 HI(5) Submitted to USDA
06-045-18n 5/18/2006 PR(3) Submitted to USDA
06-109-04n 6/14/2006 PR(3) Submitted to USDA
06-201-102n 9/11/2006 IA, PR(2) Submitted to USDA
06-033-01n 3/14/2006 IL(7), KS(5) Submitted to USDA
06-033-02n 3/23/2006 IA(7), IL(5), IN(2) Submitted to USDA
06-223-111n 9/11/2006 PR(4) Submitted to USDA
06-319-102n 12/15/2006 PR(3) Submitted to USDA

2007 07-038-117n 4/9/2007 IA(2) Submitted to USDA
07-046-108n 4/1/2007 IL(2) Submitted to USDA
07-054-103n 3/25/2007 IA, IL(2), NE, OH Submitted to USDA

07-043-104n 3/20/2007 IA(2), IL(2), IN(2), MO, NE, OH, PA, WI Submitted to USDA

07-094-105n 5/4/2007 WI Submitted to USDA

07-043-105n 4/17/2007 AR(2), IL, IN, KS, MI, MO(5) Submitted to USDA
07-137-104n 6/16/2007 PR(2) Submitted to USDA
07-247-108n 10/4/2007 PR(2) Submitted to USDA
07-211-102n 8/31/2007 PR(2) Submitted to USDA
07-254-103n 10/11/2007 IA, IL, IN, MO, NE Submitted to USDA
07-031-109n 3/21/2007 IL(10), IN(3), MO Submitted to USDA
07-031-112n 3/20/2007 AR, IA(7), KS(6), MD Submitted to USDA
07-352-101rm 3/26/2008 IA(8), IL(16), IN(4), KS(6) Submitted to USDA
07-031-102n 3/18/2007 PR(2) Submitted to USDA
07-032-101n 4/5/2007 IL(3) Submitted to USDAMonsanto Company 09-SY-201U Page 202 of 471 Table A-1 (cont.) USDA Notifications Approved for MON 87705 and Status of

Trials Conducted under These Notifications

2008 08-056-109n 3/28/2008 AR, IL(3), MD, WI Submitted to USDA

08-058-113n 3/28/2008 IA(2), IL(2), IN(2), OH, PR(2) Submitted to USDA
08-045-118n 3/15/2008 PR(2) Submitted to USDA
08-049-101n 3/19/2008 IL, MD, WI Submitted to USDA
08-058-105n 3/28/2008 IL, IN, MI, MO, WI(2) Submitted to USDA
08-080-114n 4/19/2008 IA, PR(2) Submitted to USDA
08-079-101n 4/17/2008 IA(3) Submitted to USDA
08-084-102n 4/24/2008 IA, NE Submitted to USDA

08-137-101n 6/15/2008 PR Submitted to USDA
08-170-102n 7/18/2008 PR(2) Submitted to USDA
08-182-101n 8/1/2008 PR(2) Submitted to USDA

08-270-101n 10/26/2008 PR In Progress

08-301-103n 11/26/2008 HI , PR In Progress

08-357-101rm 3/17/2009 IA(11), IL(14), KS(5), IN(3), MO, NE In Progress
08-323-101n 12/18/2008 PR(3) In Progress
09-007-106n 2/25/2009 PR(2) In Progress
08-042-101n 3/14/2008 AR, IA, IL(2), IN, MI, NE(2) Submitted to USDA
08-042-102n 3/12/2008 IA(2), IL(8), IN(4), MO Submitted to USDA
08-042-107n 3/12/2008 IA(7), KS(5) Submitted to USDA

2009

09-050-117n 3/21/2009 HI, IA(4), IL(4), IN(2), OH(2), PR, WI In Progress
09-033-101n 3/1/2009 IA(8), KS(5), NE In Progress

09-061-108n 4/1/2009 AR, IA, IL(5), IN(2), KS, MI, MO, NE, WI(2) In Progress

09-050-134n 3/21/2009 HI , PR In Progress 09-068-110n 4/8/2009 IL(5) In Progress
09-099-102n 5/9/2009 PR(2) In Progress
09-124-105n 5/13/2009 IA In Progress

09-135-103n 6/14/2009 IL In Progress

09-135-104n 6/14/2009 IL In Progress 09-030-105n 3/1/2009 IA(3), IL(4 ), IN, KS, NE In Progress
09-030-104n 3/1/2009 IA(3), IL(8 ), IN(3), MO In Progress
09-036-103n 3/7/2009 IA(2 ), IL(2), IN, MS, NE(2) In ProgressMonsanto Company 09-SY-201U Page 203 of 471 Appendix B. Materials and Methods Used for Molecular Analyses of MON 87705

B.1 Materials

The DNA used in molecular analyses was is olated from leaf tissue of MON 87705

collected in 2008 ha rvested from Production Plan 07-01-83-30 (Seed lot: GLP 0704 18620-S). Additional DNA extracted from various MON 87705 generations of leaf tissues were used in generation stabil ity analyses. The c ontrol substance was conventional soybean variety A3525 which has the same genetic background as the test

substance. The reference substance, plasmid PV-GMPQ/ HT4404 (Figures IV-1 and 2),

was used as the transformati on vector to develop MON 87705. The plasmid was digested and used as a positive hybridi zation control in Southern analyses. Probe templates generated from this plasmid also served as positive hybridization controls. As additional reference standards, the 1 kb DNA extension ladder and λ DNA/Hind III segments from Invitrogen (Carlsbad, CA) were used for si ze estimations on Southern blots and agarose gels. The 500 bp ladder from Invitrogen and GeneRuler 1 kb Plus Ladder from Fermentas (Hanover, MD) were used for size estimations for PCR analyses.

B.2 Characterization of the Materials

The quality of the source materials from MON 87705 and A3525 were verified by PCR analysis to confirm the presence or abse nce of MON 87705 except the materials used in the generational stability analyses where the id entity of the materials was confirmed by the generation stability Southe rn blots themselves. The st ability of the genomic DNA was confirmed in each Southern analysis by observation of the digested DNA sample on

an ethidium bromide- stained agarose gel.

B.3 DNA Isolation for South ern Blot and PCR Analyses

Genomic DNA from the test and control substances was extracted prior to the initiation of the study using a hexadecyltrimethyla mmonium bromide (CTAB)-based method according to SOP BR-ME-1153-01. DNA ex tractions were stored in a 4 ° C refrigerator

or a -20 ° C freezer.

B.4 Quantification of Genomic DNA

Extracted genomic DNA and plasmid DNA were quantified prior to the initiation of the study using Hoefer’s DyNA Quant 200 Fluorom eter according to SOP BR-EQ-0065-02. Molecular size marker IX (Roche, Indianap olis, IN) was used as the DNA calibration

standard.

B.5 Restriction Enzyme Digestion of Genomic DNA

Approximately 10 or 20 μ g of genomic DNA extracted from the test and control substances were used for restriction enzyme digestions. When digesting genomic DNA

with Nco I (New England BioLabs, Beverly, MA), 10X NE buffer 3 (New England BioLabs) was used. When digesting genomic DNA with the Spe I (Roche, Indianapolis, IN), 10X Tango buffer (Fermentas) was used . All digests were performed at 37 ° C according to SOP BR-ME-0316-01 in a total volume of ~500 μ l using ~25-100 units of the appropriate restriction enzyme(s).Monsanto Company 09-SY-201U Page 204 of 471 B.6 DNA Probe Preparation for Southern Blot Analyses

Probe template DNA containing sequences of plasmid PV-GMPQ/HT4404 was prepared by PCR amplification according to SOP BR- ME-0486-01 and gel purif ied according to SOP BR-ME-0889-01. The probes were desi gned based on the nu cleotide content (%GC) so that the entire probe would be hybridized under the condi tions appropriate for the sequence. Approximately 25 ng of each probe template were radiolabeled with

32 P-deoxyadenosine triphosphate (dATP) ( 6000 Ci/mmol) using the random priming method (RadPrime DNA Labeling System, I nvitrogen) according to SOP BR-ME-0611- 01. Approximately 1×10 6 cpm of labeled probe per ml of hybridization solution was hybridized to the Southern blot . Probe locations relative to the genetic elements in plasmid PV-GMPQ/HT4404 are depict ed in Figures IV-1 and IV-2.

B.7 Southern Blot An alyses of Genomic DNA

Digested genomic DNA isolated from test and control material, in addition to predigested

control material mixed with appropriate positive hybridization controls were evaluated using Southern blot analyses according to SOP BR-ME-0317-02. The plasmid DNA was digested and then added to the predigested conventional soybean geno mic DNA to serve as a positive hybridization contro l. Probe templates were a dded to predigested control

material to serve as an a dditional positive hybridization c ontrol. The DNA was then separated by agarose gel electrophoresis. S outhern blots were hybridized and washed at

55 ° C or 60 ° C depending on the calculated melting temperature (T

m ) of the probes used. The table below lists the hybridization conditi ons of the probes used in this study. Multiple exposures of each blot were then generated using Kodak Biomax MS film in conjunction with one Kodak Biomax MS intensifying screen in a -80 ° C freezer.Monsanto Company 09-SY-201U Page 205 of 471 Probe DNA Probe Element Sequence Spanned by

DNA Probe Hybridization/Wash Temperature ( ° C)

1 T-DNA I Probe 1A Left Border + P- FMV/Tsf1 + L- Tsf1 +

I- Tsf1 (portion) 55

2 T-DNA I Probe 2B I- Tsf1 (portion) + TS- CTP2 (portion) 55

3 T-DNA I Probe 3C TS- CTP2 (portion) +

CS- cp4 epsps (portion) 60

4 T-DNA I Probe 4D CS- cp4 epsps (portion) + T- E9 +

P- 7S α '

+ FAD2-1A (portion) 55

5 T-DNA I Probe 5E FAD2-1A p (portion)+ FATB1-A p (portion) + Right Border 55

6 T-DNA II Probe 1A T- H6 + FAD2-1A p + FATB1-A p 55

7 Backbone Probe 1 Backbone Sequence 60

8 Backbone Probe 2 Backbone Sequence 60

9 Backbone Probe 3 Backbone Sequence 60

10 Backbone Probe 4 Backbone Sequence 60

B.8 DNA Sequence Analyses of the Insert

Overlapping PCR products were generated th at span the insert in MON 87705. These products were sequenced to determine th e nucleotide sequence of the in sert in MON 87705, as well as determining the nuc leotide sequence of the genomic DNA flanking the 5' and 3' ends of the insert. The PCR analysis was performed according to

SOP BR-ME-0486-01.

The PCR analyses were conducted using ~ 96-100 ng of genomic DNA template or ~12 ng of plasmid DNA in a 50 μ l reaction volume or ~48-50 ng of genomic DNA template in a 25 μl reaction volume containing a final concentration of 1 mM MgSO4, 1 M
Betaine, 0.8 μ M of each primer, 0.2 mM each dNTP, and 0.02 units of KOD Hot Start DNA polymerase from Novagen (Gibbstown, NJ). The amplification of Products A, D, and E was performed under the following cycling conditions: 94 ° C for 2 minutes; 35 cycles at 94 ° C for 45 seconds, 65 ° C for 45 seconds, 72 ° C for 5 minutes; one cycle at

72 ° C for 10 minutes. The amplification of Pr oducts B, C, and F was performed under the following cycling conditions: 94 ° C for 2 minutes; 35 cycles at 94 ° C for 55 seconds,

68 ° C for 60 seconds, 72 ° C for 5 minutes; and one cycle at 72 ° C for 10 minutes.

Aliquots of each PCR product were separated on 0.8% (w/v) agarose E-gel® (Invitrogen) or separated on a 0.8% agarose gel acco rding to SOP BR-ME-0315-02. Prior to sequencing, the PCR products we re visualized by ethidium st aining to verify the products were of the expected size prior to sequenc ing. To remove excess primers following PCR amplification, products were treated with a mixture of 0.1 unit Exonuclease I, designated as EXO, from USB (Cleveland, OH) and 0.1 unit Shrimp Alkaline Phosphatase, designated as SAP (USB) per 5 μl of PCR product and cycled at the following conditions: one cycle at 37 ° C for 15 minutes, one cycle at 80 ° C for 15 minutes. As documented in the raw data, not all products were treated with EXO-SAP prior to sequencing. The PCR products were seque nced using multiple primers, including primers used for PCR amplification. In addi tion, primers internal to the amplified PCRMonsanto Company 09-SY-201U Page 206 of 471 sequences were used to sequence the regi ons of the amplicon. All sequencing was performed by the Monsanto Genomics Seque ncing Center using BigDye terminator chemistry (ABI, Foster City, CA).

B.9 PCR and DNA Sequence Analysis to Examine the MON 87705 Insertion Site

To demonstrate that the DNA sequences fla nking the insert in MON 87705 are native to the soybean genome and to examine the MON 87705 inserti on site in conventional soybean, PCR and sequence analyses were performed on genomic DNA from both MON 87705 and conventional soybean. The primer s used in this analysis were designed from the DNA sequences flanking the insert in MON 87705. One primer designed from the genomic DNA sequence flanking the 5' en d of the insert was paired with a second primer located in the genomic DNA sequence flanking the 3' end of the insert. The PCR analysis was performed acco rding to SOP BR-ME-0486-01.

The PCR analyses were conducted using ~ 96-100 ng of genomic DNA template in a 50 μl reaction volume or ~50 ng of genomic DNA template in a 25 μ l reaction volume containing a final concen tration of 1 mM MgSO4, 1 M Betaine, 0.8 μ M of each primer, 0.2 mM each dNTP, and 0.02 units of KO D Hot Start DNA polymerase (Novagen). The amplification of the product was performed under the following cycling conditions: 94°C for 2 minutes; 35 cycles at 94°C for 55 seconds, 68°C for 60 seconds, 72°C for 5 minutes; one cycle at 72°C for 10 minutes. Aliquots of each PCR product were separated on 0.8% (w/v) agarose E-gel® (Invitrogen) and visualized using the UV transilluminator to verify that the products were of the e xpected size prior to sequencing. To remove excess primer following

PCR amplification, the PCR product containing the convent ional soybean template was

treated with a mixture of 0.1 unit EXO and 0.1 unit SAP per 5 μ l of PCR product and cycled as follows: one cycle at 37°C for 15 minutes, one cycle at 80°C for 15 minutes. The PCR products were sequenced using multiple primers, including primers used for PCR amplification. In addition, primers inte rnal to the amplified PCR sequences were used to sequence the regions of the amp licon. All sequencing was performed by the Monsanto Genomics Sequencing Center usi ng BigDye terminator chemistry (ABI).Monsanto Company 09-SY-201U Page 207 of 471 Figure B-1. Overlapping PCR Analysis across the Insert in MON 87705
PCR analyses demonstrating the linkage of the individu al genetic elements within the insert in MON 87705 were performed on MON 87705 genomic DNA extracted from leaf (Lanes 3, 6, 10, 14, 17, and 20). Lanes 2, 5, 9, 13, 16, and 19 contain reactions with conventional soybean DNA while lanes 4, 8, 12, 15, 18, and 21 are reactions containing no template DNA. Lanes 7 and 11 contain reactions with PV-GMPQ/HT4404 plasmid control DNA. Lanes 1and 22 contain Fermentas GeneRuler 1 kb Plus DNA Ladder. Lanes are marked to show which product has been loaded and is visualized on the agarose gel. The expected product size for each amplicon is provided in the illustration of the insert in MON 87705 that appears near the bottom of the figure. Three to fifteen microliters of each of the PCR produc ts was loaded on the gel. This figure is representative of the data generated in th e study; however the PCR amplicons reported in this figure were not necessarily used in sequencing.

Lane 1: GeneRulerTM 1 kb Plus DNA Ladder 12: No template DNA control 2: Conventional soybean DNA 13: Conventional soybean DNA 3: MON 87705 genomic DNA 14: MON 87705 genomic DNA 4: No template DNA control 15: No template DNA control 5: Conventional soybean DNA 16: Conventional soybean DNA 6: MON 87705 genomic DNA 17: MON 87705 genomic DNA 7: PV-GMPQ/HT4404 control DNA 18: No template DNA control 8: No template DNA control 19: Conventional soybean DNA 9: Conventional soybean DNA 20: MON 87705 genomic DNA 10: MON 87705 genomic DNA 21: No template DNA control 11: PV-GMPQ/HT4404 control DNA 22: GeneRuler 1 kb Plus DNA Ladder Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

* Symbol denotes partial sequences from Left and Right Border after integration into MON 87705.Monsanto Company 09-SY-201U Page 208 of 471 Appendix C. Materials, Methods and Resu lts for Characterization of CP4 EPSPS

Protein Produced in MON 87705

C.1 Materials

The MON 87705-produced CP4 EPSPS protei n (Orion lot 10002253) was purified as

described below from harvested seed of MON 87 705 prior to the initiation of this study.The identity of the harvested seed containing MON 87705 was confirmed by eventspecific PCR; a copy of the Certificate of Analys is (COA) for this seed lot is archived in the Monsanto Regulatory archives with the records documenting protein isolation. The purified MON 87705-produced protein was stored in a -80 oC freezer in a buffer solution containing 50 mM Tris-HCl, pH 7.5, 50mM KCl, 2mM DTT, 1mM benzamidine-HCl and 25% glycerol. The reco rds describing the purificati on of this MON 87705-produced protein are archived und er the Orion lot 10002253.

The E. coli- produced CP4 EPSPS reference prot ein (Orion lot 10000739) was purified

from the fermentation of E. coli transformed with a plasmid containing the cp4 epsps gene. The DNA sequence encoding this CP 4 EPSPS reference protein was confirmed both prior to and following fermentation of E. coli . Records pertaining to the purification

of this E. coli- produced reference prot ein are archived under Orion lot 10000739. The E.

coli- produced CP4 EPSPS protein reference stan dard was previously characterized (APS Characterization Plan 20-100015) and a copy of the COA is included in the Monsanto

archives. The E. coli -produced CP4 EPSPS protein was st ored in a -80 °C freezer in a buffer solution (50 mM Tris-Cl, 1 mM ben zamidine-HCl, 50 mM KCl, 2 mM DTT, and 25% (v:v) glycerol, pH 7.5) at a total protein concentration of 3.8 mg/ml with a purity of

97%.

The E. coli- produced CP4 EPSPS protein was used as a reference protein for the immunoblot assay, the functional activity as say, and the purity and molecular weight evaluation, and as a negative contro l in the glycosylation analysis.

C.2 Description of Assay Controls

Protein molecular weight standards (BioRad, Hercules, CA) were used to calibrate SDSPAGE gels and verify prot ein transfer to PVDF memb ranes. A peptide mixture (CalMix2 from the Sequazyme Peptide Mass Sta ndards kit, Applied Biosystems, Foster City, CA) was used to calibrate the MALDI-TOF mass spectrometer for tryptic mass analysis. A PTH-amino acid standard mixture (Applied Biosystems) was used to calibrate the sequencer for N-terminal seque nce analysis. Diluti ons of BSA standard (BioRad, Hercules, CA) were used to generate a standard curve for determining total protein concentration. Tran sferrin and horseradish peroxi dase (both from Sigma, St. Louis, MO) were used as positive contro ls for glycosylation analysis. CandyCane Glycoprotein Molecular Weight Standards (Molecular Probes, Eugene, OR) were used as molecular weight markers and positive and nega tive controls for glycosylation analysis.

C.3 Protein Purification

The plant-produced CP4 EPSPS protein was purified from seed of MON 87705 prior to initiation of this characterization plan. The purification procedure was not performed under a GLP plan; however, all procedures were documented on worksheets and, whereMonsanto Company 09-SY-201U Page 209 of 471 applicable, SOPs were followed. The CP4 E PSPS protein was purifie d from an extract of ground seed using a combination of a mmonium sulfate fractionation, hydrophobic interaction chromatography, anion exchange chromatography, and cellulose phosphate affinity chromatography. All protein extraction and chromatography steps were performed at ~4°C. A detail ed description of the purifi cation process was filed under Orion Lot 10002253, and is br iefly described below.

Approximately 100 g of pre-chilled seed of MON 87705 were groun d using a Perten Laboratory Mill 3100. The ground powder (~ 100 g) was defatted 3 times with 500 ml each of Hexanes (w:v, EMD, Gibbstown, NJ) prewarmed to 37 oC, air-dried, and stored in a -80 oC freezer pr ior to extraction of the CP4 E PSPS protein. Th e portion of the defatted seed powder (50 g) was mixed with an extraction buffer (100 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM Benzamidine-HCl, 4 mM DTT, 2 mM Phenylmethylsulfonyl fluoride, 1% Polyvinylpolypyrrolidone and 10% glycerol) for 2 hours at approximately a 1:10 powder weight to extraction buffer volum e ratio. The slurry was centrifuged at

23,500 

## × g for 20 minutes at ~ 4 oC. The result ant 430 ml supernatant was subjected to 40% ammonium sulfate protei n fractionation by addition of 97 g of ammonium sulfate

over one hour in the cold room (2 oC to 8 oC). The solution was stirred for two hours at ~ 4 oC and centrifuged at 23,500 X g for 20 minutes. Another 89.8 g of ammonium sulfate was added to the supernatant (480 ml) over a period of one hour to 70%

saturation. The solution was st irred for two hours in a 4 oC co ld room and the pellet was collected by centrif ugation at 23,500 X

 g for 30 min. The pellet was resuspended in 150 ml of buffer designed as PS(A) [50 mM Tr is-HCl, pH 7.5, 1 mM DTT, 10% glycerol (v:v), 1.5 M (NH 4 )2SO 4 ]. The resuspended sample was loaded onto a 140 ml column of Phenyl Sepharose Fast Flow (5 cm X 7 cm column) (GE Healthcare, Piscataway, NJ) equilibrated with the buffer PS( A). Proteins were eluted w ith a linear salt gradient that decreased from 1.5 to 0 M (NH 4 )2SO 4 in the buffer PS(A) over a volume of 1400 ml. Fractions containing the CP4 E PSPS protein, identified base d on western blot analysis, were pooled to a final volume of ~250 ml. The pooled sample was desalted by dialysis against 4 L of buffer designed as QS(A) (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM benzamidine-HCl, 4 mM DTT) for 20 h at ~4 °C (with three addi tional buffer changes) using dialysis tubing [Molecu lar Weight Cutoff (MWCO), 12 to 14 kDa] (Spectrum Laboratories, Inc. Rancho Dominguez, CA).

The desalted sample (337 ml) was loaded onto a 60 ml column of Q Sepharose Fast Flow anion exchange resin column (2 cm X 20 cm ) (GE Healthcare, Piscataway, NJ ), which was equilibrated with the QS (A) buffer. Bound CP4 EPSPS protein was eluted with a linear salt gradient that increased from 0 M to 0.4 M potassium ch loride in the QS(A)

buffer over 600 ml. Fractions containing CP4 EPSPS protein, identified by western blot analysis, were pooled to a final volume of ~ 140 ml. The pooled sample was place into dialysis tubing (MWCO, 12 to 14 kDa, Spect rum Laboratories, Inc. Rancho Dominguez, CA) and dialyzed against buffer designed as CP(A) [50 mM MES, pH 5.8, 10% glycerol (v:v), 1 mM benzamidine-HCl and 1 mM DTT] for 18 hours at ~4 °C in three buffer changes.

The dialyzed sample (100 ml) was then lo aded onto a 19 ml cellulose phosphate P11 cation exchange column (1.6 X 9.5 cm) pre-e quilibrated with the CP(A) buffer. Bound CP4 EPSPS protein was eluted with the CP(A) buffer containing 0.5 mMMonsanto Company 09-SY-201U Page 210 of 471 phosphoenolpyruvate (PEP) and 0



### . 5 mM S3P. Fractions co ntaining CP4 EPSPS protein, based on SDS PAGE analysis , were pooled (~14.5 ml). This pooled sample was concentrated to 10 ml at ~4 °C using a slid e-A-lyzer dialysis casse tte (MWCO: 10 kDa, size: 3 to 12 ml, Pierce, Rockford, IL) and covering it in a water absorbing polymer powder (Aquacide I, EMD, Gibbstown, NJ). Af ter concentration, the cassette was placed into 2 L of dialysis buffer (50 mM Tris-H Cl, pH 7.5, 50 mM KC l, 2 mM DTT, 1 mM benzamidine-HCl) and dialyzed for a total of 20 hours at ~ 4 °C in three 2 L buffer changes. The dialyzed sample in the casse tte was further concentrated to 5 ml using Aquacide I as described above. This 5 ml sample was mixed with 5 ml dialysis buffer containing 50% glycerol to final volume of 10 ml. Fina l buffer composition of the sample was 50 mM Tris-HCl, pH 7.5, 50mM KCl, 2mM DTT, 1 mM benzamidine-HCl and 25% glycerol. This CP4 EPSPS prot ein purified from s eed of MON 87705 was aliquoted (100 μl each), assigned APS lot 10002253 and stored at ~ -80 °C.

C.4 Molecular Weight and Purity Estimation-SDS-PAGE

SDS-PAGE analysis was performed to dete rmine the molecular weight of CP4 EPSPS protein purified from MON 87705 and to compare the mo lecular weight of the MON 87705- produced and E coli -produced CP4 EPSPS proteins.

An aliquot of the test substance was mi xed with 5 X sample loading buffer (LB) [312 mM Tris-HCl, 20% (v:v ) 2-mercaptoethanol, 10% (w:v) SDS, 0.025% (w:v) bromophenol blue, 50% (v:v) glycer ol, pH 6.8)], to a final to tal protein concentration of 0.168 μg/μl. Molecular weight markers (Bio -Rad broad-range) and reference substance were diluted to a final total protein concentra tion of 0.9 and 0.2 μg/μl, respectively. The test substance was analyzed in duplicate at 1, 2, and 3 μg protein per lane. The E. coliproduced CP4 EPSPS protein reference standard (Orion lot 10000739) was analyzed at 1 μg total protein. All samples were heated at ~ 100 °C for three mi nutes and loaded onto a pre-cast Tris glycine 4 to 20% polyacrylam ide gradient 10-well mini-gel (Invitrogen, Carlsbad, CA). Electrophores is was performed at a consta nt voltage of 150 V for 95

minutes. Proteins were fixed by placing the gel in a solution of 40% (v:v) methanol and 7% (v:v) acetic acid for 30 minutes, staine d for 18 hours and 35 minutes with Brilliant Blue G-Colloidal stain (Sigma-Aldrich, St. Louis, MO), destaine d 30 seconds with a solution containing 10% (v:v) acetic acid and 25% (v:v) methanol, and finally destained with 25% (v:v) methanol for 6.5 hours. An alysis of the gel was performed using a BioRad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). Molecular weight markers were used to estimate the apparent molecular weight of each observed band. All visible bands within each lane were quantified using Quantity One software. Apparent molecula r weight and purity were reported as an average of all six lo adings containing the MON 87705- produced CP4 EPSPS protein.

C.5 Immunoblot Analysis-Immunoreactivity

Immunoblot analysis was performed to confir m the identity of the CP4 EPSPS protein purified from MON 87705 and to compare the immunorea ctivity of the MON 87705- produced and E coli -produced CP4 EPSPS proteins.

The MON 87705- produced and E. coli- produced CP4 EPSPS protei ns were both loaded onto the same gel at equal loads of 1, 2, a nd 3 ng per well. Aliquots of each protein were

diluted in water and 5 X LB heated at 100.5 °C for 3 min, and applied to a pre-cast TrisMonsanto Company 09-SY-201U Page 211 of 471 glycine 4 to 20% polyacrylamide gradient 15-we ll gel (Invitrogen, Carlsbad, CA). Three amounts of each protein were loaded in duplicate on the gel. Electrophoresis was performed at a constant voltage of 150 V fo r 88 minutes. Pre-stained molecular weight markers (SeeBlue Plus2 Prestained, Invitrogen, Carlsbad, CA) were loaded in parallel to verify electrotransfer of the proteins to the membrane and estimate the size of the

immunoreactive bands observed. Electrotransfer to a 0.45 μm nitrocellulose membrane (Invitrogen, Carlsbad, CA) was performed for 90 minutes at a constant voltage of 25 V.

For immunodetection, the membrane was bl ocked for 1 hour with 5% (w:v) Non-Fat Dried Milk (NFDM) in 1 X Phosphate Buff ered Saline containing 0.05% (v:v) Tween-20 (PBST). The membrane was then probed w ith a 1:1,000 dilution of goat anti-CP4 EPSPS antibody (lot 10000787, aliquot # 20) in 5% (w :v) NFDM in PBST fo r one hour. Excess antibody was removed using three 10 minutes washes with PBST. Finally, the membrane was probed with horseradish peroxidase (H RP)-conjugated ra bbit anti-goat IgG (Thermo, Rockford, IL) at a dilution of 1:10,000 in 5% (w:v) NFDM in PBST for one hour. Excess HRP-conjugate was removed using three 10 minu tes washes with PBST. All incubations were performed at room temperature. Immu noreactive bands were visualized using the ECL detection system (GE, Healthcare, Pisc ataway, NJ) and exposed (10, 30, and 60 s) to Amersham Hyperfilm (GE, Healthcare, Pisc ataway, NJ). Films were developed using

a Konica SRX-101A automated f ilm processor (Tokyo, Japan).

The immunoreactive bands of the MON 87705- produced CP4 EPSPS protein in each lane migrating to the same position as the referen ce standard were quantif ied and compared to the signals corresponding to the E. coli CP4 EPSPS protein reference substance. Quantification of the blot was performed us ing a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4. 0, Hercules, CA) using the lane finding and

contour tool. The raw data was exported to a Micros oft Excel [2007 (12.0.6324.5001) SP1 MSO (12.0.6320.5000] file for the pair wise comparison of all the loads. An average

absolute difference was calculated fo r each comparison to determine the immunoreactivity equivalence.

C.6 MALDI-TOF Tryptic Mass Map Analysis

MALDI-TOF MS was used to confirm th e identity of the MON 87705-produced CP4 EPSPS protein. Since the prot ein was determined to be pure (100%) based on pre-study data, it was not deemed necessary to se parate the protein by SDS-PAGE prior to trypsinization.

An ethanol precipitation was performed to concentrate the MON 87705-produced CP4 EPSPS protein sample and remove any buffer components that may interfere with the analysis. Twenty five μl of the MON 87705-produced CP4 EPSPS sample (0.21 mg/ml) was concentrated to approximately 20 μl w ith a Speed-Vac concentrator and then mixed with 200 μl prechilled 95% etha nol. After overni ght incubation at -2 0 °C, the mixture was centrifuged at 13,000 X g for 30 minutes at ~4 °C. The pellet was collected, washed twice with 200 μl of prechilled acetone and then twice with 200 μl of water. Ten μl of trypsin solution [20 μg/ml trypsin (Promega , Madison, WI) in a 25 mM ammonium bicarbonate buffer, pH 7.8] was added and incubated over night at 37 °C. Trypsin digested samples (0.3 μ l) were added directly onto the analysis plate in triplicate and followed by the addition of ~ 0.75 μl of three matrices, 2,5- dihydroxybenzoic acidMonsanto Company 09-SY-201U Page 212 of 471 (DHB), α -cyano-4-hydroxycinnamic acid ( α -Cyano) , and Sinapinic acid (Waters Corp., Milford, MA) on separate spots. The sample in DHB matrix was analyzed in the 300 to 7500 dalton range using 100 shots at a laser in tensity setting of 2480 (a unit-less MALDITOF instrument specific value) while samples in α -Cyano and Sinapinic acid were analyzed in the 500 to 7500 dalton range using 100 shots at a laser intensity setting of 1980 and 2380, respectively. Protonated (MH+) peptide masses were observed monoisotopically in reflector m ode (Aebersold, 1993; Billeci and Stults, 1993). Calmix 2 was used as the external calibrant (Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster City, CA) for the analysis. GPMAW32 software (Applied Biosystems, version 4.23) was used to genera te a theoretical trypsi n digest of the CP4 EPSPS protein sequence based upon the nucleo tide sequence. Masses were calculated for each theoretical peptide and compared to the raw mass data. Experimental masses (MH+) were assigned to peaks in the 300 to 7500 Da range if they met the following criteria: resolved monoisotopic peak; with at least one additi onal associated ion peak for

masses < 1000 Da and at least two associat ed ion peaks for masses > 1000 Da; peak height greater than twice the baseline noise ; and did not overlap with a stronger mass signal ( ± 2 daltons from the mass analyzed). Know n autocatalytic segments from trypsin digestion were identified in the raw data. The list of experimental masses was then compared to the theoretical list from the GPMAW software. Those experimental masses within one Da of a theoretical mass were matched. All matching masses were tallied and a coverage map was generated. The tr yptic mass map coverage was considered acceptable if ≥ 40 % of the protein sequence was identified by matching experimental masses observed for the tryptic peptide se gments to the expected masses for the

segments.

C.7 MALDI-TOF Mass Analysis of MO N 87705-produced CP4 EPSPS Protein

MALDI-TOF mass spectrometry was used to confirm the molecular weight of the MON 87705-produced CP4 EPSPS protein. Since the protein was determined to be pure (100%) prior to this analysis, it was not d eemed necessary to separate the protein by

SDS-PAGE.

MALDI-TOF MS was used to further char acterize the molecular weight of the MON 87705-produced CP4 EPSPS protein. Pr ior to analysis, the MON 87705-produced CP4 EPSPS protein was dialyzed using drop dialysis (Görisch , 1988). Briefly, a 25 mm Millipore microdialysis disk (type VSWP, 0. 025 μm pore size, Bedford, MA) was floated on HPLC-grade water, spotted with 2 μl of the sample, and dialyzed for 45 minutes. A portion of the MON 87705-produced CP4 EPSPS and BSA protein samples (0.125 and 0.25 μl) was spotted on an analysis plat e, mixed with 0.375 and 0.75 μl of 3,5- dimethoxy-4-hydroxycinnamic acid (Sinapinic acid ) solution, re spectively, and air-dried. Mass spectral analysis of the MON 87705-pr oduced CP4 EPSPS protein was performed using an Applied Biosystems Voyager DE -Pro Biospectrometry Workstation MALDITOF instrument with the supplie d Data Explorer software (ver sion 4.0, Foster City, CA). Mass calibration of the instrument was perf ormed using a BSA protein standard. The sample was analyzed in the 10,000 to 100,000 dalton range using 100 shots at a laser intensity setting of 2983 (a un it-less MALDI-TOF instrument specific value). Average protonated (MH+) protein masse s were observed in linear m ode (Aebersold, 1993; Billeci and Stults, 1993). GPMAW32 software (Applie d Biosystems, version 4.23, Foster City,Monsanto Company 09-SY-201U Page 213 of 471 CA) was used to generate a theoretical mass of the expected CP4 EPSPS protein sequence based upon the nucleotide sequen ce. The mass of the MON 87705-produced CP4 EPSPS protein was repor ted as an average of three separate mass spectral acquisitions.

C.8 N-Terminal Sequencing

N-terminal sequencing using automated Ed man degradation chemistry was used to confirm the identity of the M ON 87705-produced CP4 EPSPS protein.

Because the protein was determined to be 100% pure based on pre-study data, it was not necessary to separate the protein by SDS- PAGE. Therefore, an aliquot of the MON 87705-produced CP4 EPSPS pr otein was used for N-terminal sequence analysis. The analysis was performed fo r 15 cycles using automated Edman degradation chemistry (Hunkapillar et al., 1983). An Applied Bi osystems 494 Procise Sequencing System with 140C Microgradient system and 785 Progra mmable Absorbance Detector and Procise Control Software (version 1.1a) were used. Chromatographic data were collected using Atlas99 software (version 3.59a, LabSystems , Altrincham, Cheshire, England). A PTHamino acid standard mixture (Applied Bios ystems, Foster City, CA) was used to chromatographically calibrate the instrument for each analysis. This mixture served to verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times. A control protein (10 picomoles of β -lactoglobulin, Applied Biosystems, Foster City, CA) was anal yzed before and after the analysis of the CP4 EPSPS protein to verify that the sequencer met performance criteria for repetitive yield and sequence identity. Identity was deemed to be established if ≥ 8 amino acids, consistent with the predicted sequence of the N-terminus of the MON 87705-produced CP4 EPSPS protein were obs erved during analysis.

C.9 Glycosylation Analysis

Glycosylation analysis was performed to determine whether the MON 87705-produced CP4 EPSPS protein was post- translationally modified with covalently bound carbohydrate moieties. Aliquots of the MON 87705-produced CP4 EPSPS protein, the

E.-coli- produced CP4 EPSPS reference standard protein, and the positive controls, transferrin (~ 76 – 81kDa, Sigma-Aldrich, St. Louis, MO), and horsera dish peroxidase (~ 40 kDa, Pierce, Rockford, IL) were each diluted with water and mixed with 5 × LB. These samples were heated at 97.6 °C for 5 minutes, cooled, and loaded on a Tris glycine 4-20% polyacrylamide gradient 10-well mini-gel . Each sample was loaded at 50 and 100 ng per lane. SeeBlue Plus2 pre-stained prot ein molecular weight markers (Invitrogen, Carlsbad, CA) were loaded to verify electrotr ansfer of the proteins to the membrane and the CandyCane Glycoprotein Molecular Weight Standards (Molecular Probes, Eugene, OR) were loaded as positive/negative contro ls and markers for molecular weight. Electrophoresis was performed at a cons tant voltage of 150 V for 80 minutes.

Electrotransfer to a 0.45 μ m PVDF membrane (Invitrogen, Carlsbad, CA) was performed

for 90 minutes at a cons tant voltage of 25 V.

Carbohydrate detection was perf ormed directly on the PVDF membrane using the Pro-Q Emerald 488 Glycoprotein Ge l and Blot Stain Kit (M olecular Probes). The manufacturer’s protocol was followed. All steps were pe rformed at room temperature.

The PVDF membrane was fixed in 25 ml of a solution containing 50% methanol and 5%Monsanto Company 09-SY-201U Page 214 of 471 glacial acetic acid for one hour, the solution was then ch anged and the membrane was incubated overnight. Two, 15 minutes washes (50 ml each) with 3% (v:v) glacial acetic acid (wash solution) were followed by a 20 minut es oxidation in 25 ml of an oxidizing solution containing periodic acid (Component C from kit). The membrane was washed three times, 10 minutes each, in 50 ml of wash solution. The membrane was then incubated in 25 ml of Pro-Q Emerald Staini ng Solution that was prepared using the kit reagents. After 40 minutes of staining in th e dark, one 15 minutes, 50 ml wash cycle was followed by two 30 minutes, 50 ml wash cycles . The final wash cycles included two 50 ml, one minute deionized water washes fo llowed by three five minutes methanol washes(EMD, San Diego, CA). The blot was then scanned using the BioRad Molecular Imager FX using the Alexa 488 illumination setting (Quantity One software; version 4.6, build 036) in order to visualize the fluor escently labeled glycosylated proteins.

After glycosylation analysis the blot was stai ned to visualize the proteins present on the

membrane. Proteins were st ained for one minute using Coom assie Brilliant Blue R-250 staining solution (Bio-Rad, Hercules , CA) and then destained with 1 

## × destain solution (Bio-Rad, Hercules, CA) for 5 min. After washing with water, the blot was scanned using Bio-Rad GS-800 densitometer with th e supplied Quantity One software (version 4.4.0, Hercules, CA) in order to visualize total proteins.

C.10 Functional Activity Assay

In order to assess the functional activity of the MON 87705-produced CP4 EPSPS protein and to compare its activity to the E. coli- produced CP4 EPSPS reference standard protein, aliquots of the MON 87705 -produced CP4 EPSPS protein and E. coli- produced CP4 EPSPS reference standard pr otein were analyzed for their ability to release inorganic phosphate from phosphoenolpyruvate (PEP). The specific activity is expressed in units per mg of protein (U/mg), where a unit (U) is defined as 1 μ mole of inorganic phosphate released from PEP per minute at 25 ° C.

The assay was carried out on a micro titer plate. Prior to functional activity analysis, both test and reference proteins were diluted to a purity corrected concentration of 50 μ g/ml with a 50 mM HEPES, pH 7.0 buffer. Assa ys for both proteins were conducted in triplicate. Each assay replicate was subsequently analyzed spectropho tometrically in duplicate. Briefly, the reactions contai ning the CP4 EPSPS enzyme with S3P were initiated by the addition of PEP. The react ions were performed in a mixture of 50 mM HEPES (pH 7.0), 0.1 mM ammonium mol ybdate, 2 mM S3P, 1 mM PEP and 5 mM potassium fluoride for two minutes at 25.2 ° C. The reactions were quenched with malachite green (phosphate assay reagent) a nd fixed after two minut es with 33% (w:v)

sodium citrate. The releas e of inorganic phosphate from PEP was determined at a wavelength of 660 nm using a PowerWave Xi (Bio-Tek, Richmond, VA) microplate reader, and quantitated relativ e to a standard curve of i norganic phosphate treated with the malachite green (phosphate assay) reagent and 33% (w:v) sodium citrate.

C.11 Results of CP4 EPSPS Protein Molecular Weight Equivalence

The equivalence in apparent molecular we ight of the purifie d MON 87705-produced and

the E. coli- produced CP4 EPSPS protein was demons trated using SDS-PAGE (Figure C-1). The MON 87705-produced CP4 EPSPS prot ein migrated with a molecular weightMonsanto Company 09-SY-201U Page 215 of 471 indistinguishable to that of the E. coli- produced protein standard analyzed concurrently (Table C-1). Based on comparable el ectrophoretic mobilities, the MON 87705-produced

and E.coli- produced CP4 EPSPS proteins were determ ined to have equivalent apparent molecular weights.

The predicted mass of the MON 87705-produced CP4 EPSPS protein was also confirmed by MALDI-TOF MS. The average mass obtai ned for CP4 EPSPS was 47,396 Da. This experimentally obtained mass differs from the theoretical mass calculated for the CP4 EPSPS reference standard protein minus the N-terminal methionine by only 0.18%. The difference between the exp ected and the observed ma ss for MON 87705-produced CP4 EPSPS is minimal and within the acceptable error for MALDI-TOF ma ss determination.

The absence of the N-terminal methionine was confirmed by N-te rminal sequencing.

Table C-1. Molecular Weight Difference Between the MON 87705- and E. coli -
produced CP4 EPSPS Proteins.

Molecular Weight

of MON 87705-Produced

CP4 EPSPS Protein 1 Molecular Weight of E. coliproduced CP4 EPSPS Protein 2 % Difference from E. coli - produced CP4 EPSPS

Protein 3

44.6 kDa 43.8 kDa 1.8%

1 Reference Table C-1 for the molecular weight of the full-length MON 87705-produced protein.

2 Reference the Orion 10000739 COA for the molecular weight of the full-length E. coli - produced reference protein.

3 Percent difference was calculated as follows: 

### % 8

. 1 % 100 44.6 8

. 3 4 6

. 4 4

= × −Monsanto Company 09-SY-201U Page 216 of 471 Figure C-1. Molecular Weight and Puri ty Analysis of the MON 87705-Produced

CP4 EPSPS Protein Aliquots of the MON 87705- produced and the E. coli- produced CP4 EPSPS proteins were separated on a 4 to 20% Tris glycine pol yacrylamide gradient gel and then stained with Brilliant Blue G-Colloidal stain. Approximate MWs (kDa) are shown on the left and correspond to the markers loaded in Lanes 1 and 9.

Lane Sample Amount loaded ( μ g)

1 Broad Range molecular weight markers 4.5

2 E. coli - produced CP4 EPSPS protein

standard 1

3 MON 87705-produced CP4 EPSPS protein 1

4 MON 87705-produced CP4 EPSPS protein 1

5 MON 87705-produced CP4 EPSPS protein 2

6 MON 87705-produced CP4 EPSPS protein 2

7 MON 87705-produced CP4 EPSPS protein 3

8 MON 87705-produced CP4 EPSPS protein 3

9 Broad Range molecular weight markers 4.5 200

97.4
66.2

45

31

21.5

6.5 14.4 116.3

kDa

Lane # 1 2 3 4 5 6 7 8 9Monsanto Company 09-SY-201U Page 217 of 471 C.12 Results of CP4 EPSPS Protein Immunoreactivity Equivalence

A western blot analysis us ing goat anti-CP4 EPSPS serum was conducted to determine the relative immunoreactivity of the MON 87705-produced CP4 EPSPS protein and the

E. coli- produced CP4 EPSPS protein reference sta ndard. The results demonstrated that the anti-CP4 EPSPS antibody recognized the MON 87705-produced CP4 EPSPS protein that migrated to a similar position as the E. coli- produced reference standard protein (Figure C-2). Furthermore, the immunoreactive signal increased with increasing levels of CP4 EPSPS protein loaded. The observ ed immunoreactivities between the MON 87705- produced and E. coli- produced proteins were similar based on densitometric analysis of the western blot (Table C- 2). Based on the above anal ysis, the MON 87705-produced

and E. coli- produced CP4 EPSPS protei ns demonstrated equivalent immunoreactive properties, which confirmed the identity and equivalence of the two proteins.Monsanto Company 09-SY-201U Page 218 of 471 Figure C-2. Western Blot Analys is of MON 87705-produced and E. coli - produced

CP4 EPSPS Proteins

Aliquots of the purified, MON 87705-produced and E. coli- produced CP4 EPSPS proteins were separated by SDS-PAGE, and electrotransfe rred to a PVDF membrane. The membrane was probed with goat anti-C P4 EPSPS antibodies and developed using an ECL system (GE Healthcare). Approximate mo lecular weights (kDa) of markers loaded

in Lane 1 are shown on the left side of th e blot. The 30 second e xposure is shown. *: Non-assigned molecular weight in marker.

Lane Sample Amount

Loaded

(ng)

1 See Blue® Plus2 Pre-Stained molecular weight markers -

2 E. coli - produced CP4 EPSPS reference standard 1

3 E. coli - produced CP4 EPSPS reference standard 1

4 E. coli- produced CP4 EPSPS reference standard 2

5 E. coli - produced CP4 EPSPS reference standard 2

6 E. coli - produced CP4 EPSPS reference standard 3

7 E. coli - produced CP4 EPSPS reference standard 3 8 Empty 9 MON 87705-produced CP4 EPSPS protein 1 10 MON 87705-produced CP4 EPSPS protein 1 11 MON 87705-produced CP4 EPSPS protein 2 12 MON 87705-produced CP4 EPSPS protein 2 13 MON 87705-produced CP4 EPSPS protein 3 14 MON 87705-produced CP4 EPSPS protein 3 250

148

98

64

50

34

17

16

7

4 * Lane # 1 2 3 4 5 6 7 8 9 10 11 12 13 14

kDa

CP4 EPSPSMonsanto Company 09-SY-201U Page 219 of 471 Table C-2. Comparison of Immunoreactive Signal between MON 87705-produced

and E. coli - produced CP4 EPSPS Proteins

Sample Gel

lane Amount (ng) Density (OD x

mm 2 ) Average Density 1 Percent

Difference 2

(%) Average

Difference 3

(%)

E. coli CP4 EPSPS 2 1 0.994 0.957 9.8 10.5 ± 1.8

E. coli CP4 EPSPS 3 1 0.920

MON 87705 CP4 EPSPS 9 1 1.027 1.062

MON 87705 CP4 EPSPS 10 1 1.096

E. coli CP4 EPSPS 4 2 1.904 2.163 13.0

E. coli CP4 EPSPS 5 2 2.421

MON 87705 CP4 EPSPS 11 2 2.584 2.485

MON 87705 CP4 EPSPS 12 2 2.386

E. coli CP4 EPSPS 6 3 3.296 3.766 8.7

E. coli CP4 EPSPS 7 3 4.236

MON 87705 CP4 EPSPS 13 3 4.039 4.124

MON 87705 CP4 EPSPS 14 3 4.208

1 Average Density = ∑ 〖 Density 〗 /2

2 Percent Difference ሺ%ሻ ൌ |A୴ୣ୰ୟ୥ୣ Dୣ୬ୱ୧୲୷ ୮୪ୟ୬୲ିA୴ୣ୰ୟ୥ୣ Dୣ୬ୱ୧୲୷ E.ୡ୭୪୧|

A୴ୣ୰ୟ୥ୣ Dୣ୬ୱ୧୲୷ ୮୪ୟ୬୲ X 100%

3 Average difference (%) = ∑ 〖 % difference 〗 /3. The standard deviation was calculated using Microsoft Office Excel 2007 (12.0.6324.5001) SP1 MSO (12.0.6320.5000).Monsanto Company 09-SY-201U Page 220 of 471 C.13 Results of MALDI-TOF Tryptic Mass Map Analysis

The MON 87705-produced, CP4 EPSPS protei n was assessed by MALDI-TOF MS. Prior to analysis, the protein sample was chem ically reduced, alkylated , and digested with trypsin. The ability to identify a protein us ing this method is dependent on matching a sufficient number of observed mass segments to expected (theoretical) mass segments. In general, protein id entification made by peptide mapping is considered to be reliable if the measured coverage of the sequence is 15% or higher with a minimum of five matched peptides. Observed tryptic peptides were c onsidered a match to th e expected tryptic mass when differences in molecular weight of less than one dalton (D a) were found between the observed and predicted segment masses. Such matches were made without consideration for potential natural amino acid modifications such as glycosylation. The protein sample was digested with trypsin a nd the masses of the tr yptic peptides were

measured.

There were 30 unique peptide segments (Table C-3), identified that corresponded to the expected masses of the CP4 EPSPS trypsin-digested peptides. The identified masses were used to assemble a coverage map indi cating the matched peptide sequences for the entire CP4 EPSPS protein (Fi gure C-3), resulting in ~80% (362 out of 455 amino acids) coverage of the total protein. This anal ysis confirmed the identity of the MON 87705- produced CP4 EPSPS protein.Monsanto Company 09-SY-201UPage 221 of 471Table C-3. Summary of the Tryptic Masses Identified forthe MON 87705-Produced CP4 EPSPS Using MALDI-TOF MS.MatrixExpectedMass1Difference2AAposition3Segmentα-CyanoDHB Sinapinicacid599.31616.32629.32629.32711.45835.39863.46872.45872.45948.52991.561115.581357.731359.671359.671646.861705.821994.032183.242367.43389.18416.23474.20506.17599.27616.29629.28629.28711.42835.37863.44872.43872.43948.501115.581357.731359.691359.691558.901646.891705.881994.072183.302367.503186.353249.77616.10835.291359.541359.541558.731993.822183.052367.212450.132450.133186.303249.464188.82389.250.07 225-227 TIR416.30474.27506.22599.33616.34629.29629.34711.45835.39863.46872.45872.52948.52991.551115.571357.711359.721359.641558.831646.841705.810.070.070.050.020.020.030.0200000.0700.010.010.020.050.030.070.020.0170-72228-231354-35729-33128-132201-205383-388133-13862-6915-23313-320358-366161-16814-23295-305146-157354-36634-4647-61389-405367-382IRKLEGRESDRSISHRRPMGRDHTEKGRPDGKVLNPLRAMQAMGARSSGLSGTVRGVTVPEDRLSAVANGLKTPTPITYRKSSGLSGTVRLAGGEDVADLRSEDGDRLPVTLRESDRLSAVANGLKSFMFGGLASGETRITGLLEGEDVINTGKGLGNASGAAVATHLDHRLNGVDCDEGETSLVVR1993.972183.172367.332450.232450.223186.523249.624188.260.060.070.100.100.090.170.150.56206-224275-294178-20024-46105-12773-104321-351234-274MLQGFGANLTVETDADGVRTGLILTLQEMGADIEVINPRSAVLLAGLNTPGITTVIEPIMTRIPGDKSISHRSFMFGGLASGETRLTMGLVGVYDFDSTFIGDASLTKEGDTWIIDGVGNGGLLAPEAPLDFGNAATGCRAPSMIDEYPILAVAAAFAEGATVMNGLEELRLTGQVIDVPGDPSSTAFPLVAALLVPGSDVTILNVLMNPTR1Only experimental masses that matched expected masses are listed in the table.2The numbers represent the difference between the expected mass and the first column which has the corresponding numbers.3AA position refers to amino acid position within the predicted CP4 EPSPS sequence as depicted in Figure C-3.Monsanto Company 09-SY-201U Page 222 of 471 .

001 MLHGASSRPA TARKSSGLSG TVRIPGDKSI SHRSFMFGGL ASGETRITGL

051 LEGEDVINTG KAMQAMGARI RKEGDTWIID GVGNGGLLAP EAPLDFGNAA

101 TGCRLTMGLV GVYDFDSTFI GDASLTKRPM GRVLNPLREM GVQVKSEDGD

151 RLPVTLRGPK TPTPITYRVP MASAQVKSAV LLAGLNTPGI TTVIEPIMTR

201 DHTEKMLQGF GANLTVETDA DGVRTIRLEG RGKLTGQVID VPGDPSSTAF

251 PLVAALLVPG SDVTILNVLM NPTRTGLILT LQEMGADIEV INPRLAGGED

301 VADLRVRSST LKGVTVPEDR APSMIDEYPI LAVAAAFAEG ATVMNGLEEL

351 RVKESDRLSA VANGLKLNGV DCDEGETSLV VRGRPDGKGL GNASGAAVAT

401 HLDHRIAMSF LVMGLVSENP VTVDDATMIA TSFPEFMDLM AGLGAKIELS

451 DTKAA

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87705-produced CP4

EPSPS.

The amino acid sequence of the CP4 EPSPS protein was deduced from the cp4 epsps
gene present in MON 87705. Boxed regions correspond to tryptic pe ptides that were
identified from the MON 87705-produced CP4 EPSPS protein sample using MALDITOF MS. In total, 79.6% (362 of 455 total am ino acids) of the expe cted protein sequence

was identified.Monsanto Company 09-SY-201U Page 223 of 471 C.14 Results of N-terminal Sequence Analysis

N-terminal sequencing of the first 10 am ino acids performed on MON 87705-produced CP4 EPSPS protein resulted in the sequence expected for the CP4 EPSPS protein (Table C-4) with the exception of the N-terminal methionine, which was not detected. This result is expected as removal of the N-te rminal methionine, catalyzed by methionine aminopeptidase, is a common modification that occurs co-translationally before completion of the nascent protein chain and has no effect on protein stru cture or activity. The N-terminal sequence information, therefore, confirms the identity of the CP4 EPSPS protein isolated from MON 87705 and the intactness of its N-terminus.

Table C-4. N-Terminal Sequence of the MON 87705-produced CP4 EPSPS.

Amino acid

residue # from

the N-terminus → 1 2 3 4 5 6 7 8 9 10 11

Expected Sequence 1 → M L H G A S S R P A T

│ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence → - L H G A S S R P A T

The expected amino acid sequence of the N- terminus of the CP4 EPSPS protein was

deduced from the cp4 epsps gene present in soybean MON 87705. The experimental sequence obtained from the MON 87705-produc ed CP4 EPSPS was compared to the expected sequence. 1 The single letter IUPAC-IUB amino acid code is M , methionine; L

, Leucine; H, histidine; G , glycine; A , alanine, S , serine; R , Arginine; P , proline; T , threonine; and (-) Indicates the M residue not observed.

C.15 Results of Glycosylation Analysis

Many eukaryotic proteins are post-translat ionally modified with carbohydrate moieties (Rademacher et al., 1988). These carbohydrat e moieties may be complex, branched polysaccharide structures or simple monos accharides. In contrast, strains of E. coli used for recombinant protein expression lack the n ecessary biochemical pathways required for protein glycosylation. To assess whether poten tial post-translational glycosylation of the MON 87705-produced CP4 EPSPS protein occurred, the purif ied protein sample was subjected to glycosyl ation analysis. The E. coli- produced CP4 EPSPS reference standard represented a negative control. The positive controls were the transferrin and horseradish peroxidase (HRP) proteins which are know n to have multiple covalently linked carbohydrate modifications. The transferrin prot ein and HRP, as well as the purified CP4 EPSPS protein isolated from MON 87705 and E. coli were separated on SDS-PAGE,Monsanto Company 09-SY-201U Page 224 of 471 transferred to a PVDF membra ne, and glycosylation analysis was performed to detect carbohydrate moieties on the proteins. The result s of this analysis are shown in Figure C4. The positive controls, transferrin and HRP, were detected at th e expected molecular weights of ~75 and ~50 kDa, respectively, in a concentration-dependent manner (Figure C-4, Panel A, Lanes 4-5 and 2-3). A very faint signal, slightly above background, was observed for the MON 87705-produced and E. coli- produced CP4 EPSPS proteins (Figure C-4, Panel A, Lanes 6-7 and 8-9). This low level sign al could be due to low level oxidation of amino acid residues of the prot ein and/or nonspecific binding of florescent reagents. Further evidence that the si gnals observed for MON 87705-produced and

E. coli -produced CP4 EPSPS proteins are nonspeci fic are the following: (1) The faint signal was also associated with the E. coli- produced CP4 EPSPS could not be the product of a glycan moiety because E. coli- produced proteins are not glycosylated. (2) Mass spectrometry data demonstr ated the absence of glyc osylation of the MON 87705- produced CP4 EPSPS. Glycosyl ation would result in an in crease in the protein mass relative to the theoretically ca lculated mass. No increase in protein mass was observed for the MON 87705-produced CP4 EPSPS protein determined by MALDI-TOF MS (47396 Da) as compared to its theoreti cal mass (47481.48 Da) (3 ) Four potential glycosylation sites can be identified in the amino aci d sequence of the CP4 EPSPS protein: one O linked at T248 and three N-linked at N 213, N271 and N392 (see Figure C3 for amino acid positions). The tryptic se gments containing these amino acids were identified for the MON 87705-produced CP4 EPSPS protein by MALDI-TOF mass spectrometry. All identified masses matche d the expected non-modified peptide masses, indicating that no glycosyl ation had occurred.

To confirm that suffici ent MON 87705-produced and E. coli- produced CP4 EPSPS proteins were present for carbohydrate detection and gl ycosylation analysis, the

membrane was stained with Coomassie Blue R 250 stain to detect pr oteins (Figure C-4, Panel B). Both MON 87705-produced and E. coli -produced CP4 EPSPS were clearly detected on the membrane (Figure C-4, Panel B, Lanes 6-9).

These results indicate th at the MON 87705-produced CP 4 EPSPS protein is not glycosylated and, thus is equivalent to the E. coli- produced CP4 EPSPS reference

standard.Monsanto Company 09-SY-201U Page 225 of 471 Figure C- 4. Glycosylation Analysis of the MON 87705-Produced CP4 EPSPS

Protein

Aliquots of the MON 87705-produc ed CP4 EPSPS protein, E. coli- produced CP4 EPSPS reference standard (negative control), hors eradish peroxidase (p ositive control) and transferrin (positive control) were separated by SDS-PAGE (4

- 20% gradient) and electrotransferred to a PVDF membrane. ( A ) Where present, peri odate-oxidized proteinbound carbohydrate moieties reacted with Pr o-Q Emerald 488 glyc oprotein stain and emitted a fluorescent signal at 488 nm (Lanes 1-5). ( B ) The same blot was stained with Coomassie Blue R250. The signal was cap tured using a Bio-Rad GS-800 scanner. Approximate molecular weights (kDa) co rrespond to the CandyCane glycosylated

markers loaded in Lane 1

Lane Sample Amount (ng)

1 CandyCane Glycoprotein mo lecular weight standards

⎯ 2 Horseradish Peroxidase (positive control) 50 3 Horseradish Peroxidase (positive control) 100 4 Transferrin (positive control) 50 5 Transferrin (positive control) 100 6 MON 87705-produced CP4 EPSPS 50 7 MON 87705-produced CP4 EPSPS 100

8 E. coli- produced CP4 EPSPS (negative control) 50

9 E. coli- produced CP4 EPSPS (negative control) 100

10 See Blue® Plus2 Pre-Staine d molecular weight markers

⎯ Lane # 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 

### A B

180

42 82

18 CP4 EPSPSMonsanto Company 09-SY-201U Page 226 of 471 C.16 Results of Functional Activity

The functional activities of the MON 87705-produced and E. coli -produced CP4 EPSPS proteins were estimated usi ng an assay that measures th e EPSPS-catalyzed formation of inorganic phosphate (P i ) and 5-enolpyruvylshikimat e-3-phosphate (EPSP) from Shikimate-3-phosphate (S3P) and Phosphoenolpyr uvate (PEP). In this assay protein specific activity is expressed as units per m illigram of protein (U /mg), where a unit is

defined as one μ mole of inorganic phosphate rele ased from PEP per minute at 25 ° C. The MON 87705-produced and E. coli -produced CP4 EPSPS prot eins were considered functionally equivalent if th e specific activity of one pr otein was within 50% (assay variability) of the other.

Results indicated that the specific activity was 4.10 U/mg protein for the MON 87705- produced CP4 EPSPS, and 4.38 U/mg protein for the E. coli- produced reference standard. The difference in specific activities was 6.4% (Table C-5) which is within assay variability. These resu lts clearly demonstrat e that the CP4 EPSPS pr oteins derived from

MON 87705 and E. coli have equivalent f unctional activities.

Table C-5. CP4 EPSPS Functional Assay

The specific activity of the plant-produced CP4 EPSPS protein was determined using a phosphate release assay. This end-point type colorimetric assay meas ures the release of inorganic phosphate from one of the substr ates, PEP, by the action of the CP4 EPSPS

enzyme.

MON 87705- produced

CP4 EPSPS 1
(U/mg) E. coli- produced

CP4 EPSPS 1
(U/mg) Difference (%, Plant vs E. coli) 2

4.10 ± 0.1 4.38 ± 0.33 6.4

1 Value refers to mean and standard deviation calculated based on n= 6

2 % Difference ൌ ሺAୡ୲୧୴୧୲୷ ா.௖௢௟௜ିAୡ୲୧୴୧୲୷ MON ଼଻଻଴ହሻ

Aୡ୲୧୴୧୲୷ ா.௖௢௟௜ 

## × 100%Monsanto Company 09-SY-201U Page 227 of 471 Appendix C References

Aebersold, R. 1993. Mass spectrometry of proteins and peptid es in biotechnology. Current Opininons in Biotechnology 4:412-419.

Billeci, T.M., and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrixassisted laser desorption/ionization mass spectrometry. Analytical Chemistry 65:1709-

1716.

Gorisch, H. 1988. Drop Dialysis: Time Course of Salt and Protein Exchange. Analytical Biochemistry 173:393-398.

Hunkapillar, M.W., R.M. Hewick, W.J. Drey er, and L.E. Hood. 1983. High-sensitivity sequencing with gas-phase seque nator. Methods in Enzymology. 91:399-413.

Rademacher, T.W., R.B. Parekh, and R.A. Dwek. 1988. Glycobiology. Annual Review in Biochemistry 57:785-838.Monsanto Company 09-SY-201U Page 228 of 471 Appendix D. Materials and Methods Used for the Analysis of the

Levels of CP4 EPSPS Protein in MON 87705

D.1 Materials

Tissue samples analyzed in this study were prod uced from five field sites in Chile during the 2007-2008 planting season from seed lot GLP-0702-18254-S for MON 87705 and

GLP-0702-18252-S for the conventional control. The conventional control, A3525 , does

not contain the cp4 epsps coding region. Samples were stored in a -80 ° C freezer throughout the study. An E. coli- produced CP4 EPSPS protein (Monsanto APS lot 20-

100015 ) was used as a reference standard for the assay.

D.2 Characterization of the Materials

The identities of the MON 87705 and conventional control substances were confirmed by analysis of the harvested seed DNA by an event-specific polymerase chain reaction (PCR) method and the resulting Verifications of Identity (VOIs) were archived in the Monsanto Regulatory Archives. The copies of VOIs were also included in the study file.

D.3 Field Design and Tissue Collection

Production Plan REG-07-170 was initiated during the 2007-2008 pl anting season to generate MON 87705 and conventional control substances at various soybean-growing

locations in Chile. The field sites were as follows: Quilapilum, Chacabuco; Melipilla, Melipilla; Calera de Tango, Maipo; Rancagua , Cachapoal; and San Fernando, Colchagua. These field sites were representative of soybean producing regions suitable for commercial soybean production. At each site, three replicated plots of soybean plants containing MON 87705 and the conventional c ontrol were planted using a randomized complete block field design. Throughout th e field production sa mple identity was maintained by using unique sample iden tifiers and proper chain-of-custody documentation. All tissue samples, except s eed, were stored in a -80 oC freezer and shipped on dry ice to the Monsanto processing facility in St. Louis, Missouri. Seed samples were stored and shi pped at ambient temperature.

Over-season leaf tissue sample s were collected from the younge st set of fully expanded trifoliate leaves at the following growth stag es: OSL1 at V2-V3 gr owth stage; OSL2 at V7; OSL3 at V10; and OSL4 at V14. The root and forage tissues were collected at approximately the R5-R6 growth stage, a nd the above-ground porti on of the plant was labelled as the forage, and the below ground portion was washed and labelled as root tissue. Harvested seed samples were collected at the R8 growth stage.Monsanto Company 09-SY-201U Page 229 of 471 D.4 Tissue Processing and Protein Extraction

All tissue samples produced at the field site s were shipped to M onsanto’s processing facility. During the processing step, dry ice was combined with the individual samples,

and vertical cutters or mixe rs were used to thoroughly grind and mix the tissues. Processed samples were transferred into capped 50 ml tubes and stored in a -80 ° C freezer

until use.

The CP4 EPSPS protein was extrac ted from soybean tissues as described in Table D-1. The CP4 EPSPS protein was extracted from all le af, forage, root and seed tissues using a Harbil Mixer with the appropriate amount of Tris-borate buffer with L-ascorbic acid (TBA) [0.1 M Tris, 0.1 M Na 2 B 4 O 7 · 10H 2 O, 0.01 M MgCl 2 , 0.05% (v:v) Tween-20 at pH 7.8, and 0.2% (w:v) L-ascorbic acid]. Insoluble material was removed from all tissue extracts using a serum filter (Fisher Scient ific, Pittsburgh, PA). The extracts were aliquotted and stored frozen in a -80 ° C freezer until ELISA analysis.

Table D-1. Protein Extraction Methods for Tissue Samples

Tissue Extraction

Buffer T:B

Ratio No. of 1⁄4 in

Chrome-steel

beads Shake

time (minutes) Sample

Clarification

method

Leaf 1 TBA 1:50 8 3.5 Serum Filter

Root TBA 1:50 8 3.5 Serum Filter

Forage TBA 1:50 8 3.5 Serum Filter

Seed TBA 1:50 8 3.5 Serum Filter

1 Over-season leaf (OSL-1, OSL-2, OSL-3, and OSL-4).

D.5 CP4 EPSPS Antibodies

The capture monoclonal antibody (Lot 10002190) was specific for the CP4 EPSPS protein. The concentration of the purified antibody was determined to be 2.3 mg/ml by spectrophotometric methods. The detection re agent was goat anti-CP4 EPSPS polyclonal antibodies (Sigma, St. Louis, MO) conjuga ted to horseradish peroxidase (HRP).

D.6 CP4 EPSPS ELISA Method

The CP4 EPSPS ELISA was performed manually according to an in-house SOP. Mouse anti-CP4 EPSPS antibody was diluted in coating buffer (15 mM Na 2 CO 3 , 35 mM

NaHCO 3 , and 150 mM NaCl, pH 9.6) and immobilized onto 96-we ll microtiter plates at

2.0 μ g/ml by dispensing 100 μ l per well, followed by incubation in a 4 ° C refrigerator for greater than 12 hours. Plates were washed three times with 1X phos phate buffered saline (PBS) with 0.05% (v:v) Tween-20 (1X PBST) followed by the addition of 100 μ l per well of CP4 EPSPS protein standard or sample extract and incubated at 37 ° C for one

hour. Plates were washed three times w ith 1X PBST, followed by the addition of 100 μ l per well of goat anti-CP4 EPSPS peroxi dase conjugate and incubated at 37 ° C for one

hour. Plates were washed 3 times w ith 1X PBST, and developed by adding 100 μ l per well of HRP substrate, 3,3',5,5'- tetram ethylbenzidine (TMB, Kirkegaard & Perry,Monsanto Company 09-SY-201U Page 230 of 471 Gaithersburg, MD). The enzymatic reac tion was terminated by the addition of 100 μ l per

well of 6 M H 3 PO 4 after a 10 minute development. Quantitation of CP4 EPSPS protein was accomplished by interpolation from a CP4 E PSPS protein standard curve that ranged in concentration from 0.456 – 14.6 ng/ml.

D.7 Moisture Analysis

All tissues were analyzed fo r moisture content using an IR-200 Moisture Analyzer (Denver Instrument Company, Arvada, CO). A homogeneous tissue-specific site pool (TSSP) was prepared using the test and contro l samples of a given tissue type grown at a given site. These pools were prepared for all tissues in this study. The average percent moisture for each TSSP was calculated from triplicate analyses. A TSSP Dry Weight Conversion Factor (DWCF) was calculated as follows:

DWCF = 1 – [Mean % TSSP Moisture / 100]

The DWCF was used to convert protein levels assessed on a μg/g fres h weight (fwt) basis into levels reported on a μg/g dry weight (dwt) basis usi ng the following calculation:



## ( ) ( ) DWCF Weight Fresh Level Protein Weight Dry in Level Protein =

The protein levels that were reported to be le ss than or equal to the LOD or less than the LOQ on a fresh weight basis were no t reported on a dry weight basis.

D.8 Data Analyses

The CP4 EPSPS ELISA plates were anal yzed on a SPECTRAmax Plus (Molecular Devices, Sunnyvale, CA) microplate spec trophotometer, using a dual wavelength detection method. All protein concentrations were determined by optical absorbance at a wavelength of 450 nm with a simultaneous refe rence reading of 620 nm . Data reduction analyses were performed using Molecula r Devices SOFTmax PRO GxP version 5.0.1 software. Absorbance readings and protein standard concentrations were fitted with a four-parameter logistic curve fit. Following the interpolation from the standard curve, the amount of protein (ng/ml) in th e tissue was converted to a “ μ g/g fwt” basis. For all proteins, this conversion utiliz ed a sample dilution factor a nd a tissue-to-buffer ratio. The protein values in “ μ g/g fwt” were also converted to “ μ g/g dwt” by applying the DWCF. Microsoft Excel 2007 (12.0.6324.5001) SP1 MSO (12.0.6320.5000) (Microsoft, Redmond, WA) was used to calculate the CP 4 EPSPS protein levels in soybean tissues.Monsanto Company 09-SY-201U Page 231 of 471 Appendix E. Materials and Methods Us ed for Compositional Analysis of MON 87705 Soybean Seed, Forage and Processed Fractions

E.1 Materials

MON 87705, a conventional soybean contro l (A3525), and conventional commercial reference soybean varieties were grown at five locations in Chile in the 2007-2008 growing season. MON 87705 and the conventiona l control were grown from seed lots GLP-0702-18254-S and GLP-0702-18252-S, respectiv ely. The conventional control material, A3525, has background genetics re presentative of MON 87705 but does not

contain FATB1-A and FAD2-1A gene segments. Samples from all three replicates of test, control, and reference plot s were analyzed with the exce ption of a single replicate of the control substance from site QUI, whic h was lost. In addition, 20 commercial conventional soybean varieties were produ ced alongside of MON 87705 and 19 of these were analyzed for generation of a 99% to lerance interval. On e commercial soybean variety had all replicates da maged by an early frost and wa s excluded from the study (see Section E.3 below). The 19 varieties, locati ons, and seed lot numbers are listed below:

Material Name Seed Lot Number Site Code

Asgrow A2869 GLP-0707-18806-S SFR

Stine 2788 GLP-0707-18832-S SFR

Asgrow A3244 GLP-0707-18807-S SFR

Hoegemeyer 333 GLP-0707-18823-S SFR

NK 32Z3 GLP-0707-18827-S CdT

Stine 3300-0 GLP-0707-18833-S CdT

Channel Bio 3461 GLP-0707-18816-S CdT

Stewart 3454 GLP-0707-18831-S CdT

Croplan 3596STS GLP-0707-18818-S MEL

Garst 3585N GLP-0707-18820-S MEL

Pioneer 93B52 GLP-0707-18829-S MEL
Quality Plus 365C GLP-0707-18830-S MEL

Stine 3600-0 GLP-0707-18834-S QUI

Channel Bio 37002 GLP-0707-18817-S QUI

Lewis 372 GLP-0707-18825-S QUI

Pioneer 93B82 GLP-0707-18828-S QUI

Lewis 391 GLP-0707-18826-S RAN

Stine 3870-0 GLP-0707-18835-S RAN

Asgrow A4324 GLP-0707-18809-S RAN

E.2. Characterization of the Materials

The identities of the forage and seed sa mples from MON 87705, conventional control, and reference soybean varieties were verified prior to their use in the study by confirmingMonsanto Company 09-SY-201U Page 232 of 471 the chain-of-custody documentation supplied with the forage and seed collected from the field plots. The presence or absence of FATB1-A and FAD2-1A gene segments in the seed of MON 87705, conventional control and reference soybean varieties was verified by event-specific polymerase chain reaction (P CR). As expected, the results confirm that MON 87705 contains these gene segments a nd the conventional control and reference

varieties do not.

E.3. Field Production of the Samples

A total of 20 different conventional commercia l soybean reference va rieties were planted

at five field locations with f our different varieties grown at each site. Seeds were planted in a randomized complete block design with three replicates per block for each test, control, and reference variety. Samples from all three replicates of test, control, and reference plots were analyzed, with the exce ption of a single replicate of the control variety from site QUI, which was lost in trans it from the field location to its destination in the U.S. All replicates of the reference variety UA 4805 from s ite RAN were damaged by an early frost and were ex cluded from this study. All the samples were grown under normal agronomic field conditions for their respective geographic regions.

E.4. Summary of Analytical Methods

Harvested soybean seed and forage sample s from MON 87705, the conventional control and conventional reference soybean varie ties were shipped on dry ice to Covance Laboratories Inc. (Madison, WI) for compositi onal analyses. Analyses were performed using methods that are currently used to eval uate the nutritional quality of food and feed.

E.4.1. Acid Detergent Fiber

The sample was placed in a fritted vessel a nd washed with an acidic boiling detergent solution that dissolved the protein, carbohydrat e, and ash. An acetone wash removed the fats and pigments. The lignocellulose fracti on was collected on the frit and determined gravimetrically. The limit of quantitation was 0.100%.

Literature Reference USDA. 1970. Forage and Fiber Analyses. Ag riculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

E.4.2. Amino Acid Composition

The following 18 amino acids were analyzed:

• Total aspartic acid (i ncluding asparagine)

• Total threonine

• Total serine

• Total glutamic acid (i ncluding glutamine)

• Total proline

• Total glycine

• Total alanineMonsanto Company 09-SY-201U Page 233 of 471 • Total valine

• Total isoleucine

• Total leucine

• Total tyrosine

• Total phenylalanine

• Total histidine

• Total lysine

• Total arginine

• Total tryptophan

• Total methionine - (sulfur containing)

• Total cystine (including cyst eine) - (sulfur containing)

The sample was assayed by three methods to obtain the full profile. Tryptophan required a base hydrolysis with sodium hydroxide. Th e sulfur-containing ami no acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids wa s accomplished through direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantitated using an automate d amino acid analyzer. The limit of quantitation was 0.100 mg/g.

Reference Standards

• ThermoScientific K18, 2.5 μ mol/mL per constituent except cystine (1.25

μ mol/mL), Lot Number JC120602

• Sigma, L-Tryptophan, 100%, Lot Number 076K50075

• Sigma/BioChemika, L-Cysteic Acid M onohydrate, >99% (used as 100%), Lot

Number 1305674

• Sigma, L-Methionine Sulfone, 100%, Lot Number 012H3349

Literature Reference

AOAC. 2005. Method 982.30 in Of ficial Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.3. Ash

The sample was placed in an electric furnace at 550 ° C and ignited to drive off all volatile organic matter. The nonvolatile matter re maining was quantitated gravimetrically and calculated to determine pe rcent ash. The limit of quantitation was 0.100%.

Literature Reference

AOAC. 2005. Method 923.03 in Of ficial Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.4. Carbohydrates

The total carbohydrate level was calculated by difference usin g the fresh weight-derived data and the following equation:Monsanto Company 09-SY-201U Page 234 of 471 % carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The limit of quantitation was 0.100%.

Literature Reference USDA. 1973. Energy value of foods. Agri culture Handbook No. 74. United States Department of Agriculture, Washington, D.C.

E.4.5. Fat by Acid Hydrolysis

The sample was hydrolyzed with hydrochloric ac id at an elevated temperature. The fat

was extracted with ether and hexane. The extract was evaporated on a steambath, redissolved in hexane and filtered through a s odium sulfate column. The hexane extract was then evaporated again on a steambath under nitrogen, dried, and weighed. The limit of quantitation was 0.100%.

Literature Reference

AOAC. 2005. Methods 922.06 a nd 954.02 in Official Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.6. Fat by Soxhlet Extraction

The sample was weighed into a cellulose thim ble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the sample to remove the fat. The extract was then evaporated, dried, and weighed. The limit of quantitation was 0.100%.

Literature Reference

AOAC. 2005. Method 960.39 in Of ficial Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.7. Fatty Acid Profile with Trans Fat by GC

The lipid was extracted, sa ponified with 0.5 N methan olic sodium hydroxide, and methylated with 14% BF3-methanol. The result ing methyl esters of the fatty acids were extracted with heptane. An internal standard was added prior to the lipid extraction. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation. The limit of quantitation was 0.0200%.

Reference Standards

• Nu Chek Prep GLC Reference Standard Hazelton No. 1*, Lot Number AU18-S

• Nu Chek Prep GLC Reference Standard Hazelton No. 2*, Lot Number M13-O

• Nu Chek Prep GLC Reference Standard Hazelton No. 3*, Lot Number MA18-S

• Nu Chek Prep GLC Reference Standard Hazelton No. 4*, Lot Number AU18-S (*Overall purity of the sum of the mixture of components is 100%)

• Nu Chek Prep Methyl Gamma Linolenate, used as 100%, Lot Number U-63M-JY12-

RMonsanto Company 09-SY-201U Page 235 of 471 • Nu Chek Prep Methyl Tridecanoate, used as 100%, Lot Number N-13M-A2-S

• Nu Chek Prep Methyl Butyrate, used as 100%, Lot Number N-4M-J20-R

• Nu Chek Prep Methyl Hexanoate, us ed as 100%, Lot Number N-6M-A25-R

• Nu Chek Prep Methyl Erucate, us ed as 100%, Lot Number U-79M-AU3-Q

• Nu Chek Prep Methyl Lignocerate, us ed as 100%, Lot Number N-24M-AU18-S

• Nu Chek Prep Methyl Docosapentaenoa te, used as 100%, Lot Number U-101MJY14-S

• Nu Chek Prep Methyl Docosahexaenoate , used as 100%, Lot Number U-84M-M30-S

• Nu Chek Prep Methyl Eicosapentaenoate , used as 100%, Lot Number U-99M-D14-R

• Cayman Chemicals Steariodonic Acid Met hyl Ester, used as 100%, Lot Number

182102-1

• Nu Chek Prep Methyl Elaidate, us ed as 100%, Lot Number U-47M-JA18-R

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-F27-R

• Nu Chek Prep Methyl Nervonate, us ed as 100%, Lot Number U-88M-AU4-S

• Nu Chek Prep Methyl Palmitelaidate, used as as 100%, Lot Number U-41M-M21-S

• Monsanto Mono Trans SDA, used as 99%, Lot Numb er GLP-0804-19309-A

• Monsanto Mono Trans Alpha Linolenic Acid, used as 100%, Lot Number GLP-0804-

19308-A

• Monsanto 9c, 15c Octadecadienoate (Omn isoy), used as 100%, Lot Number GLP0802-19168-A

• Larodan Methyl 6(z), 9(z)-Octadecadienoa te, used as 99.3%, Lot Number LX-017

• Monsanto Omnisoy C17:1 Met hyl 9-cis-Heptadecenoate, us ed as 99%, Lot Number

GLP-0806-19436-A

Literature References

AOAC. 2005. Method 996.06 in Of ficial Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

AOCS. 1997. Method Ce 2c-66 and Ce 1C-89 in Official Methods and Recommended

Practices of the AOCS, 5th ed. American Oil Chemists' Society, Champaign, IL.

AOAC. 2005. Method 983.23 in Of ficial Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD

E.4.8. Isoflavone Analysis

The sample was extracted using a solution of hydrochloric acid and reagent alcohol heated on steam baths or hot plates. The ex tract was brought to volume, diluted, and centrifuged. An aliquot of the supernatan t was placed onto a C18 solid-phase extraction column. Unwanted components of the matrix were rinsed off with 20% methanol and

then the isoflavones were eluted with 80% methanol. The sample was analyzed on a high-performance liquid chromatography (HPLC) system with ultrav iolet detection and was compared to an external standard curv e of known standards fo r quantitation. The limit of quantitation was 10.0 ppm for each component.Monsanto Company 09-SY-201U Page 236 of 471 Reference Standards

Chromadex, Daidzein, 96.5%, Lot Number 04007-120

Chromadex, Glycitein, 96.3%, Lot Number 07344-571

Indofine, Genistein, >99% 1 , Lot Number 0309074

1 Used as 100% in calculations.

Literature References

Seo, A. and C.V. Morr. 1984. Improved high-performance liquid chromatographic analysis of phenolic acids a nd isoflavonoids from soybean protein products. Journal of Agricultural and Food Ch emistry, 32(3):530-533.

Pettersson, H., and K.H. Kie ssling. 1984. Liquid Chromatogr aphic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official Analytical Chemists Journal, 67(3):503-506.

E.4.9. Lectin

The sample was suspended in phosphate buffered saline (PBS), shaken, and filtered. An aliquot of the resulting extract was serially diluted in 10 cuvettes c ontaining PBS. A 10% hematocrit of lyophilized rabbi t blood in PBS was added to each dilution. After 2.5 hours, the absorbance of each dilution of the sa mple and lectin control was measured on a spectrophotometer at 620 nm, using PBS to ze ro the instrument. One hemagglutinating unit (H.U.) was defined as the level that caus ed 50% of the standard cell suspension to

sediment in 2.5 hours. The limit of quantitation was 0.10 H.U./mg.

Reference Standard

Sigma-Aldrich, Red Blood Cells, Rabb it, Product #R1629, Lot Number 105K6042.

Literature References

Klurfeld, D. M. and Kritchevsky, D. 1987. Isolation and quantitation of lectins from vegetable oils. Lipids 22:667-668.

Klurfeld, D. M., Personal communication.

Liener, I. E., 1955. The photometric determin ation of the hemagglu tinating activity of soyin and crude soybean extracts. Arch ives of Biochemistry and Biophysics 54:223-231,

E.4.10. Moisture

The sample was dried in a v acuum oven at approximately 100 ° C to a constant weight. The moisture weight loss was determined a nd converted to percent moisture. The limit of quantitation was 0.100%.Monsanto Company 09-SY-201U Page 237 of 471 Literature Reference

Official Methods of Anal ysis of AOAC INTERNATIONAL

, 18 th Ed., Methods 926.08 and 925.09, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005).

E.4.11. Neutral Detergent Fiber, Enzyme Method

The sample was placed in a fritted vessel and washed with a neutral boiling detergent solution that dissolved the protein, carbohydr ate, enzyme, and ash. An acetone wash removed the fats and pigments. Hemicellu lose, cellulose, and lignin fractions were

collected on the frit and de termined gravimetrically. The limit of quantitation was

0.100%.

Literature References

Approved Methods of the American Associ ation of Cereal Chemists, 9th Ed., Method 32.20, (1998).

USDA. 1970. Forage and Fiber Analyses. Ag riculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

E.4.12. Phytic Acid

The sample was extracted using 0.5 M HCl with ultrasonication. Purification and concentration were accomplished on a silica- based anion-exchange column. The sample was analyzed on a polymer HPLC column PRP-1, 5 μ m (150 x 4.1 mm) with a refractive index detector. The limit of quantitation was 0.100%.

Reference Standard

Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 95%, Lot Number 077K0693.

Literature References

Lehrfeld, Jacob, “HPLC Separation and Quantit ation of Phytic Acid and Some Inositol Phosphates in Foods: Problem and Soluti ons,” Journal of Agricultural and Food Chemistry, 42:2726-2731, (1994).

Lehrfeld, Jacob, “High-Performance Liquid Ch romatography Analysis of Phytic Acid on a pH-Stable, Macroporous Polymer Column,” Cereal Chemistry, 66(6):510-515, (1989).

E.4.13. Protein

Nitrogenous compounds in the sample were re duced in the presence of boiling sulfuric acid and a mercury catalyst mixture to form am monia. The acid digest was made alkaline.

The ammonia was distilled and then titrated with a previously standardized acid. The percent nitrogen was calculated and converted to equivalent protein us ing the factor 6.25. The limit of quantitation was 0.100%.Monsanto Company 09-SY-201U Page 238 of 471 Literature References

Official Methods of Anal ysis of AOAC INTERNATIONAL

, 18 th Ed., Methods 955.04 and 979.09, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005).

Bradstreet, R. B., The Kjeldahl Method fo r Organic Nitrogen, Academic Press: New York, New York, (1965).

Kolthoff, I. M., and Sandell, E. B., Quan titative Inorganic Analysis, MacMillan: New York, (1948).

E.4.14. Raffinose and Stachyose The sample was extracted with deionize d water and the extract treated with a hydroxylamine hydrochloride solution in pyridine, containing phenyl- β -D-glucoside as an internal standard. The resulting oxime s were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization det ector. The quantitation limit was 0.0500%.

Reference Standards

• Sigma, Raffinose Pentahydrate, 99% (84% after correction for degree of hydration),

Lot Number 037K1059

• Sigma, Stachyose, 98% (96.8% after corre ction for degree of hydration), Lot

Number 038K3775

Literature References

Brobst, K. M. 1972. Gas-Liquid Chromatogr aphy of Trimethylsilyl Derivatives in Methods in Carbohydrate Chemistry, Vol. 6. Academic Press, New York.

Mason, B.S., and H.T. Slover. 1971. A Gas Chromatographic Method for the Determination of Sugars in Foods. Journa l of Agricultural and Food Chemistry, 19(3):551-554.

E.4.15. Trypsin Inhibitor

The sample was ground and defatted with petr oleum ether. A sample of matrix was extracted with 0.01 N sodium hydroxide. Vary ing aliquots of the sample suspension were exposed to a known amount of trypsin and benzoy1-DL-arginine~p~nitroanilide hydrochloride. The sample was allowed to react for 10 minutes at 37°C. After 10 minutes, the reaction was halted by the addi tion of acetic acid. The solution was centrifuged, then the absorban ce was determined at 410 nm. Trypsin inhibitor activity was determined by photometrically measuring th e inhibition of trypsin’s reaction with benzoyl-DL-arginine~p~nitroani lide hydrochloride. The lim it of quantitation was 1.00 Trypsin Inhibitor Units (TIU)/mg.

Literature Reference

AOCS. 1997. Method Ba 12-75 in Official Methods and Reco mmended Practices of the American Oil Chemists’ Society. AOCS Press, Champaign, IL.Monsanto Company 09-SY-201U Page 239 of 471 E.4.16. Vitamin E

The sample was saponified to break down any fat and release vitamin E. The saponified mixture was extracted with ethyl ether and then quantitated by hi gh-performance liquid chromatography using a silica column. Th e limit of quantitation was 0.500 mg/100 g.

Reference Standard

USP, Alpha Tocopherol, 100%, Lot Number M.

Literature References

Cort, W.M., T.S. Vincente, E.H. Waysek, a nd B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high-performance liquid chromatographic Fluorescence. Journal of Agricultural Food Chemistry, 31:1330-1333.

McMurray, C.H., W.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction techniques on determination of α -tocopherol in animal feed stuffs. Journal of the

Association of Official Analytical Chemists

, 63(6):1258-1261.

Speek, A.J., J. Schijver, and W.H.P. Schreurs . 1985. Vitamin E composition of some seed oils as determined by high-performance liquid chromatography with fluorometric quantitation. Journal of Food Science, 50(1):121-124.Monsanto Company 09-SY-201U Page 240 of 471 E.5. Data Processing and Statistical Analysis

After compositional analyses were performed at Covance Laboratories, Inc., data spreadsheets were forwarded to Monsanto Comp any. The data were reviewed, formatted, and sent to Certus International, Inc. for statistical analysis.

The following formulas were used for re-exp ression of composition da ta for statistical analysis:

Component From (X) To Formula 1

Proximates (excluding Moisture), Fiber, Phytic Acid, Raffinose, Stachyose % FW % DW X/d

Amino Acids (AA) mg/g FW % DW X/(10d) Isoflavones μg/g FW μg/g DW X/d Trypsin Inhibitor TIU/mg FW TIU/mg DW X/d Vitamin E mg/100g FW mg/100g DW X/d Lectin H.U./mg FW H.U./mg DW X/d

Fatty Acids (FA) % FW % Total FA (100)X j / Σ X,

for each FA j where Σ X is

over all the FA

1 ‘X’ is the individual samp le value; ‘d’ is the fraction of the sample that is dry matter.

In order to complete a statis tical analysis for a compositional component , at least 50% of the values for a component had to be greate r than the assay limit of quantitation (LOQ). Components with more than 50% of observa tions below the assay LOQ were excluded from summaries and analysis as there was in sufficient data to conduct the statistical analysis. The following 17 components with more than 50% of the observations below the assay LOQ were excluded: 8:0 caprylic ac id, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristolei c acid, 15:0 pentadecylic acid , 15:1 10c pentadecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid , 17:1 9c heptadecenoic acid, 18:1 9t octadecenoic acid, 18:2 9c,15c octadecadienoic acid, 18:2 6c,9c oc tadecadienoic acid, 18:3 gamma linolenic acid, 20:2 11c,14c eicosadienoic acid, 20:3 11c,14c,17c eicosatrienoic acid, and 20 :4 arachidonic acid.

Otherwise, results below the LOQ were assi gned a value equal to one half of the quantitation limit. Thirteen observations fo r 24:0 lignoceric acid were assigned a value, equal to one half of the quantitation limit (<0.0200%).

PRESS residuals were used to identify outli ers. A PRESS residual is the difference between any value and its predic ted value from a statistical model that excludes the data point. The studentized version scales these re siduals so that the values tend to have a

standard normal distribution when outliers are absent. Thus, mo st values are expected to be between ±3. Extreme data points that we re outside the ±6 studentized PRESS residual range were considered for exclusion from the final analyses. No results had PRESS

residual values outside of the ±6 range.Monsanto Company 09-SY-201U Page 241 of 471 Soybean components were statistically anal yzed using a mixed model analysis of variance. The five replicated sites were analyzed both separately and combined. Individual replicated site analyses used model (1).

(1) Yij = U + Ti + Bj + eij,

where Yij = unique indi vidual observation, U = overall mean , Ti = substance effect, Bj = random block effect, and e ij = residual error.

Combined-site analyses used model (2).

(2) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk,

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, Lj = random location effect, B(L)jk = random bl ock within location effect, LTij = random location by substance interaction eff ect, and eijk = residual error.

For each compositional component, 99% tolerance intervals were calculated. A tolerance

interval is an interval that one can claim, with a specified degree of confidence, contains at least a specified proportion, p, of an entire sampled population for the parameter

measured. The calculated tolerance intervals inthis study are expected to contain, with 95% confidence, 99% of the quantities expresse d in the population of conventional soybean. Each tolerance interval estimate was base d upon the average of three observations per unique reference substance. Because negative quantities are not possible, negative calculated lower to lerance bounds were set to zero.

SAS software was used to generate all summary statistics and perform all analyses. Report tables present p-values from SAS as either <0.001 or the actual value truncated to three decimal places.Monsanto Company 09-SY-201UPage 242 of 471Table E-1. Statistical Summary of Site CdT Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Acid Detergent Fiber32.08 (3.03)27.25 (3.03)4.83 (3.78)-11.44, 21.090.329(23.18 - 42.11)[30.53 - 34.11][19.21 - 33.03][1.02 - 12.39][18.29, 41.02]Ash8.74 (0.36)8.53 (0.36)0.22 (0.51)-1.97, 2.400.710(6.76 - 10.40)[8.40 - 9.19][7.76 - 9.32][-0.68 - 1.43][6.78, 9.91]Carbohydrates72.60 (0.75)73.03 (0.75)-0.43 (1.05)-4.96, 4.110.724(63.74 - 80.60)[72.28 - 72.79][70.97 - 74.33][-1.61 - 1.82][64.45, 80.50]Moisture (% FW)71.53 (0.58)71.17 (0.58)0.37 (0.83)-3.19, 3.920.700(65.80 - 82.00)[70.60 - 72.50][70.00 - 72.10][-1.50 - 2.50][62.26, 83.45]Neutral Detergent Fiber37.26 (1.43)32.98 (1.43)4.28 (1.89)-3.86, 12.420.151(24.70 - 46.55)[34.35 - 41.05][32.26 - 33.67][2.10 - 8.05][22.57, 46.52]Protein13.34 (0.51)12.97 (0.51)0.37 (0.72)-2.74, 3.470.662(9.51 - 19.93)[13.05 - 13.89][11.85 - 14.16][-1.10 - 2.04][7.38, 21.27]Monsanto Company 09-SY-201UPage 243 of 471Table E-1. Statistical Summary of Site CdT SoybeanForage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (%DW)Total Fat5.32 (0.47)5.52 (0.47)-0.20 (0.67)-3.07, 2.660.788(1.19 - 8.22)[4.77 - 5.71][4.40 - 6.47][-1.70 - 1.31][0, 9.74]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were setto zero.Monsanto Company 09-SY-201UPage 244 of 471Table E-2. Statistical Summary of SiteCdT Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitamin, Antinutrientand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.53 (0.017)1.56 (0.017)-0.023 (0.012)-0.077, 0.0300.200(1.34 - 1.78)[1.51 - 1.54][1.51 - 1.58][-0.041 - 0.00038][1.25, 1.92]Arginine2.55 (0.036)2.55 (0.036)-0.0053 (0.040)-0.18, 0.160.905(2.15 - 3.23)[2.48 - 2.63][2.51 - 2.58][-0.062 - 0.071][1.81, 3.62]Aspartic Acid3.84 (0.045)3.94 (0.045)-0.10 (0.035)-0.25, 0.0510.104(3.37 - 4.76)[3.76 - 3.93][3.86 - 4.00][-0.16 - -0.039][3.02, 5.11]Cystine0.62 (0.0081)0.62 (0.0081)0.0014 (0.011)-0.048, 0.0510.911(0.53 - 0.64)[0.60 - 0.63][0.60 - 0.63][-0.027 - 0.028][0.49, 0.69]Glutamic Acid5.99 (0.083)6.17 (0.083)-0.19 (0.070)-0.49, 0.110.115(5.14 - 7.73)[5.86 - 6.13][6.00 - 6.29][-0.32 - -0.094][4.42, 8.48]Glycine1.52 (0.016)1.55 (0.016)-0.027 (0.0087)-0.064, 0.0100.089(1.30 - 1.79)[1.50 - 1.54][1.51 - 1.57][-0.040 - -0.011][1.19, 1.95]Monsanto Company 09-SY-201UPage 245 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Histidine0.94 (0.012)0.94 (0.012)0.0024 (0.014)-0.056, 0.0610.875(0.79 - 1.07)[0.92 - 0.97][0.92 - 0.96][-0.021 - 0.025][0.74, 1.16]Isoleucine1.61 (0.039)1.64 (0.039)-0.034 (0.038)-0.20, 0.130.460(1.37 - 2.00)[1.57 - 1.64][1.55 - 1.71][-0.11 - 0.022][1.23, 2.15]Leucine2.56 (0.031)2.63 (0.031)-0.073 (0.030)-0.20, 0.0570.137(2.26 - 3.14)[2.53 - 2.59][2.55 - 2.69][-0.13 - -0.026][2.06, 3.41]Lysine2.31 (0.026)2.32 (0.026)-0.013 (0.019)-0.096, 0.0710.581(2.00 - 2.63)[2.27 - 2.34][2.26 - 2.36][-0.051 - 0.012][1.87, 2.81]Methionine0.53 (0.0091)0.54 (0.0091)-0.0089 (0.013)-0.064, 0.0460.560(0.46 - 0.59)[0.51 - 0.54][0.52 - 0.55][-0.035 - 0.024][0.43, 0.63]Phenylalanine1.71 (0.024)1.76 (0.024)-0.050 (0.024)-0.15, 0.0530.172(1.50 - 2.11)[1.68 - 1.74][1.70 - 1.80][-0.097 - -0.025][1.35, 2.31]Monsanto Company 09-SY-201UPage 246 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Proline1.65 (0.026)1.68 (0.026)-0.027 (0.011)-0.075, 0.0210.135(1.43 - 2.03)[1.60 - 1.70][1.65 - 1.73][-0.046 - -0.0075][1.29, 2.21]Serine1.69 (0.069)1.71 (0.069)-0.013 (0.097)-0.43, 0.400.905(1.55 - 2.05)[1.59 - 1.80][1.56 - 1.81][-0.22 - 0.24][1.44, 2.15]Threonine1.28 (0.034)1.33 (0.034)-0.050 (0.048)-0.26, 0.160.406(1.19 - 1.48)[1.25 - 1.34][1.26 - 1.39][-0.13 - 0.080][1.12, 1.53]Tryptophan0.40 (0.0088)0.42 (0.0088)-0.017 (0.0061)-0.043, 0.00940.109(0.33 - 0.48)[0.38 - 0.41][0.41 - 0.43][-0.028 - -0.0070][0.30, 0.50]Tyrosine1.22 (0.021)1.17 (0.021)0.048 (0.029)-0.079, 0.170.247(1.07 - 1.39)[1.18 - 1.27][1.15 - 1.19][-0.0086 - 0.12][0.99, 1.49]Valine1.72 (0.042)1.74 (0.042)-0.023 (0.049)-0.24, 0.190.684(1.45 - 2.13)[1.69 - 1.75][1.63 - 1.82][-0.11 - 0.065][1.31, 2.29]Monsanto Company 09-SY-201UPage 247 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)16:0 Palmitic2.31 (0.040)10.80 (0.040)-8.49 (0.047)-8.70, -8.29<0.001(8.78 - 11.51)[2.29 - 2.32][10.72 - 10.91][-8.59 - -8.43][7.62, 12.55]18:0 Stearic3.17 (0.040)4.58 (0.040)-1.42 (0.038)-1.58, -1.25<0.001(3.82 - 7.21)[3.09 - 3.23][4.53 - 4.66][-1.47 - -1.34][2.87, 7.15]18:1 Oleic76.44 (0.19)23.02 (0.19)53.42 (0.21)52.50, 54.34<0.001(20.77 - 27.19)[76.35 - 76.60][22.51 - 23.37][53.19 - 53.85][18.40, 30.22]18:2 Linoleic10.09 (0.15)52.43 (0.15)-42.34 (0.11)-42.83, -41.84<0.001(48.62 - 54.74)[9.94 - 10.22][52.13 - 52.78][-42.56 - -42.19][47.75, 56.46]18:3 Linolenic6.90 (0.047)8.15 (0.047)-1.25 (0.044)-1.44, -1.060.001(5.89 - 9.11)[6.85 - 6.94][8.08 - 8.28][-1.33 - -1.18][4.97, 9.93]20:0 Arachidic0.29 (0.0058)0.35 (0.0058)-0.063 (0.0082)-0.099, -0.0280.016(0.28 - 0.54)[0.28 - 0.29][0.34 - 0.36][-0.082 - -0.046][0.22, 0.53]Monsanto Company 09-SY-201UPage 248 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic0.36 (0.0056)0.21 (0.0056)0.16 (0.0045)0.14, 0.18<0.001(0.15 - 0.22)[0.36 - 0.38][0.19 - 0.21][0.15 - 0.16][0.13, 0.25]22:0 Behenic0.28 (0.0046)0.30 (0.0046)-0.016 (0.0066)-0.045, 0.0120.131(0.29 - 0.46)[0.28 - 0.29][0.29 - 0.31][-0.029 - -0.0044][0.22, 0.47]24:0 Lignoceric0.15 (0.0029)0.15 (0.0029)-0.0049 (0.00046)-0.0069, -0.00300.008(0.056 - 0.21)[0.14 - 0.15][0.15 - 0.16][-0.0055 - -0.0040][0.030, 0.26]Fiber (% DW)Acid Detergent Fiber18.23 (0.32)16.27 (0.32)1.97 (0.45)0.014, 3.920.049(12.46 - 21.25)[17.57 - 18.58][15.69 - 16.74][0.83 - 2.89][12.71, 19.29]Neutral Detergent Fiber21.04 (0.45)17.99 (0.45)3.05 (0.30)1.78, 4.330.009(12.25 - 20.89)[20.47 - 22.18][17.71 - 18.54][2.75 - 3.65][12.07, 21.51]Proximate (% DW)Ash6.37 (0.093)6.22 (0.093)0.15 (0.099)-0.28, 0.570.274(5.64 - 6.82)[6.13 - 6.54][6.18 - 6.31][-0.050 - 0.26][5.26, 7.17]Monsanto Company 09-SY-201UPage 249 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Proximate (% DW)Carbohydrates41.82 (0.20)40.05 (0.20)1.76 (0.23)0.78, 2.750.016(32.79 - 42.29)[41.62 - 42.00][39.64 - 40.52][1.31 - 2.00][30.78, 45.86]Moisture (% FW)10.80 (0.35)10.87 (0.35)-0.067 (0.50)-2.21, 2.080.905(6.89 - 12.50)[10.10 - 11.20][10.20 - 11.40][-1.30 - 0.90][5.51, 13.37]Protein33.75 (0.33)34.22 (0.33)-0.47 (0.36)-2.02, 1.080.320(29.51 - 40.25)[33.26 - 34.65][34.04 - 34.41][-0.94 - 0.24][26.12, 43.51]Total Fat18.09 (0.25)19.52 (0.25)-1.43 (0.35)-2.95, 0.0830.055(16.91 - 23.48)[17.66 - 18.35][18.96 - 19.82][-2.16 - -0.61][15.35, 25.95]Vitamin (mg/100g DW)Vitamin E3.34 (0.13)4.03 (0.13)-0.69 (0.18)-1.46, 0.0680.059(1.09 - 5.10)[3.17 - 3.50][3.74 - 4.19][-1.01 - -0.39][0, 7.36]AntinutrientLectin (H.U./mg DW)1.93 (0.74)3.12 (0.74)-1.19 (1.01)-5.54, 3.150.359(0.65 - 8.10)[1.01 - 2.77][2.20 - 4.93][-2.93 - 0.57][0, 6.44]Monsanto Company 09-SY-201UPage 250 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Antinutrient (% DW)Phytic Acid1.81 (0.10)1.79 (0.10)0.017 (0.14)-0.59, 0.620.914(1.42 - 2.27)[1.52 - 1.97][1.78 - 1.81][-0.26 - 0.16][1.35, 2.35]Raffinose0.51 (0.018)0.52 (0.018)-0.0095 (0.025)-0.12, 0.0990.741(0.40 - 0.80)[0.50 - 0.54][0.50 - 0.56][-0.069 - 0.030][0.27, 0.87]Stachyose3.76 (0.15)3.10 (0.15)0.66 (0.15)0.027, 1.290.046(2.30 - 4.53)[3.55 - 4.16][3.04 - 3.21][0.51 - 0.95][1.96, 4.41]Trypsin Inhibitor (TIU/mg DW)42.86 (3.21)43.65 (3.21)-0.79 (2.92)-13.37, 11.800.812(23.11 - 60.42)[38.51 - 48.03][37.36 - 49.78][-5.31 - 4.69][8.75, 63.43]Isoflavone (μg/g DW)Daidzein1192.24 (52.19)1182.58 (52.19)9.66 (73.81)-307.91, 327.230.907(320.54 - 3061.22)[1145.72 - 1259.84][1092.43 - 1309.26][-163.54 - 167.41][0, 3328.03]Genistein813.17 (33.64)825.33 (33.64)-12.17 (47.57)-216.84, 192.500.822(433.41 - 2301.59)[809.79 - 815.32][751.67 - 914.22][-104.43 - 62.73][0, 2727.33]Monsanto Company 09-SY-201UPage 251 of 471Table E-2 (continued). Statistical Summaryof Site CdT Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Glycitein140.40 (18.28)146.58 (18.28)-6.18 (25.85)-117.41, 105.050.833(21.67 - 354.30)[90.89 - 176.80][141.57 - 152.56][-61.67 - 35.23][0, 376.03]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were setto zero.Monsanto Company 09-SY-201UPage 252 of 471Table E-3. Statistical Summary of Site MEL Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Acid Detergent Fiber27.77 (1.88)27.46 (1.88)0.31 (1.52)-6.24, 6.860.858(23.18 - 42.11)[24.18 - 29.83][25.10 - 31.32][-1.49 - 3.34][18.29, 41.02]Ash8.58 (0.33)8.20 (0.33)0.38 (0.46)-1.61, 2.380.497(6.76 - 10.40)[8.32 - 8.86][7.66 - 9.08][-0.52 - 1.01][6.78, 9.91]Carbohydrates71.69 (1.41)71.00 (1.41)0.69 (2.00)-7.91, 9.280.764(63.74 - 80.60)[69.52 - 73.15][67.88 - 73.56][-2.05 - 5.27][64.45, 80.50]Moisture (% FW)70.43 (0.26)69.90 (0.26)0.53 (0.37)-1.06, 2.130.287(65.80 - 82.00)[70.20 - 70.80][69.40 - 70.50][-0.20 - 1.40][62.26, 83.45]Neutral Detergent Fiber30.89 (1.70)32.58 (1.70)-1.69 (2.21)-11.22, 7.840.524(24.70 - 46.55)[27.64 - 35.57][32.19 - 33.01][-4.90 - 2.56][22.57, 46.52]Protein13.95 (1.60)14.11 (1.60)-0.16 (2.27)-9.90, 9.590.951(9.51 - 19.93)[12.65 - 16.40][11.63 - 17.85][-5.20 - 3.56][7.38, 21.27]Monsanto Company 09-SY-201UPage 253 of 471Table E-3 (continued). Statistical Summary of Site MEL Soybean Forage Fiber and Proximate Content for MON 87705 vs.the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Total Fat5.79 (0.31)6.76 (0.31)-0.97 (0.17)-1.71, -0.220.030(1.19 - 8.22)[5.37 - 6.57][6.49 - 7.19][-1.16 - -0.62][0, 9.74]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were setto zero.Monsanto Company 09-SY-201UPage 254 of 471Table E-4. Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitamin, Antinutrientand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.62 (0.032)1.56 (0.032)0.052 (0.040)-0.12, 0.230.323(1.34 - 1.78)[1.54 - 1.68][1.54 - 1.61][-0.00088 - 0.13][1.25, 1.92]Arginine2.78 (0.097)2.63 (0.097)0.15 (0.12)-0.37, 0.660.341(2.15 - 3.23)[2.57 - 2.94][2.54 - 2.79][0.025 - 0.39][1.81, 3.62]Aspartic Acid4.14 (0.12)3.94 (0.12)0.19 (0.16)-0.51, 0.900.354(3.37 - 4.76)[3.87 - 4.36][3.84 - 4.15][0.026 - 0.52][3.02, 5.11]Cystine0.61 (0.013)0.58 (0.013)0.030 (0.018)-0.048, 0.110.239(0.53 - 0.64)[0.60 - 0.62][0.55 - 0.61][-0.0014 - 0.062][0.49, 0.69]Glutamic Acid6.59 (0.23)6.18 (0.23)0.41 (0.31)-0.90, 1.730.308(5.14 - 7.73)[6.09 - 7.02][5.99 - 6.55][0.098 - 1.02][4.42, 8.48]Glycine1.61 (0.042)1.55 (0.042)0.060 (0.053)-0.17, 0.290.374(1.30 - 1.79)[1.52 - 1.69][1.52 - 1.62][-0.00070 - 0.17][1.19, 1.95]Monsanto Company 09-SY-201UPage 255 of 471Table E-4 (continued). Statistical Summaryof Site MEL Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Histidine0.96 (0.025)0.91 (0.025)0.047 (0.033)-0.096, 0.190.295(0.79 - 1.07)[0.91 - 1.01][0.89 - 0.95][0.011 - 0.11][0.74, 1.16]Isoleucine1.74 (0.052)1.62 (0.052)0.12 (0.073)-0.19, 0.440.232(1.37 - 2.00)[1.61 - 1.84][1.58 - 1.66][-0.0014 - 0.26][1.23, 2.15]Leucine2.78 (0.072)2.65 (0.072)0.13 (0.096)-0.28, 0.540.308(2.26 - 3.14)[2.62 - 2.92][2.60 - 2.76][0.024 - 0.32][2.06, 3.41]Lysine2.41 (0.049)2.31 (0.049)0.10 (0.069)-0.19, 0.400.278(2.00 - 2.63)[2.31 - 2.50][2.25 - 2.39][0.016 - 0.25][1.87, 2.81]Methionine0.56 (0.0097)0.52 (0.0097)0.038 (0.013)-0.018, 0.0940.100(0.46 - 0.59)[0.53 - 0.57][0.51 - 0.53][0.013 - 0.056][0.43, 0.63]Phenylalanine1.85 (0.053)1.78 (0.053)0.070 (0.074)-0.25, 0.390.445(1.50 - 2.11)[1.74 - 1.95][1.73 - 1.87][-0.0084 - 0.22][1.35, 2.31]Monsanto Company 09-SY-201UPage 256 of 471Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed AminoAcid, Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Proline1.81 (0.056)1.68 (0.056)0.13 (0.070)-0.18, 0.430.213(1.43 - 2.03)[1.69 - 1.89][1.63 - 1.78][0.043 - 0.27][1.29, 2.21]Serine1.79 (0.046)1.79 (0.046)-0.0013 (0.065)-0.28, 0.280.985(1.55 - 2.05)[1.72 - 1.86][1.73 - 1.89][-0.17 - 0.12][1.44, 2.15]Threonine1.34 (0.042)1.37 (0.042)-0.025 (0.053)-0.25, 0.200.676(1.19 - 1.48)[1.31 - 1.39][1.28 - 1.47][-0.13 - 0.031][1.12, 1.53]Tryptophan0.41 (0.0088)0.41 (0.0088)-0.0023 (0.012)-0.056, 0.0510.870(0.33 - 0.48)[0.39 - 0.43][0.40 - 0.42][-0.028 - 0.019][0.30, 0.50]Tyrosine1.25 (0.023)1.24 (0.023)0.0014 (0.027)-0.12, 0.120.963(1.07 - 1.39)[1.23 - 1.26][1.20 - 1.30][-0.040 - 0.054][0.99, 1.49]Valine1.86 (0.054)1.73 (0.054)0.13 (0.077)-0.20, 0.460.221(1.45 - 2.13)[1.72 - 1.96][1.68 - 1.76][-0.014 - 0.28][1.31, 2.29]Monsanto Company 09-SY-201UPage 257 of 471Table E-4 (continued). Statistical Summaryof Site MEL Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)16:0 Palmitic2.39 (0.021)10.83 (0.021)-8.44 (0.029)-8.56, -8.31<0.001(8.78 - 11.51)[2.35 - 2.42][10.80 - 10.87][-8.47 - -8.38][7.62, 12.55]18:0 Stearic3.33 (0.10)4.39 (0.10)-1.06 (0.15)-1.69, -0.420.018(3.82 - 7.21)[3.20 - 3.47][4.26 - 4.64][-1.32 - -0.79][2.87, 7.15]18:1 Oleic76.10 (0.22)22.31 (0.22)53.79 (0.31)52.45, 55.14<0.001(20.77 - 27.19)[75.68 - 76.33][21.86 - 22.61][53.06 - 54.47][18.40, 30.22]18:2 Linoleic10.50 (0.22)53.48 (0.22)-42.98 (0.31)-44.31, -41.66<0.001(48.62 - 54.74)[10.16 - 10.92][53.22 - 53.89][-43.74 - -42.40][47.75, 56.46]18:3 Linolenic6.58 (0.055)8.00 (0.055)-1.42 (0.076)-1.74, -1.090.002(5.89 - 9.11)[6.53 - 6.65][7.86 - 8.10][-1.56 - -1.31][4.97, 9.93]20:0 Arachidic0.30 (0.0058)0.34 (0.0058)-0.040 (0.0066)-0.068, -0.0120.026(0.28 - 0.54)[0.29 - 0.30][0.32 - 0.34][-0.052 - -0.030][0.22, 0.53]Monsanto Company 09-SY-201UPage 258 of 471Table E-4 (continued). Statistical Summaryof Site MEL Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic0.35 (0.0050)0.20 (0.0050)0.14 (0.0063)0.12, 0.170.001(0.15 - 0.22)[0.34 - 0.36][0.20 - 0.21][0.14 - 0.16][0.13, 0.25]22:0 Behenic0.30 (0.0063)0.30 (0.0063)0.0014 (0.0089)-0.037, 0.0400.891(0.29 - 0.46)[0.29 - 0.30][0.28 - 0.31][-0.016 - 0.020][0.22, 0.47]24:0 Lignoceric0.15 (0.0080)0.16 (0.0080)-0.0026 (0.0096)-0.044, 0.0390.812(0.056 - 0.21)[0.14 - 0.17][0.15 - 0.16][-0.019 - 0.014][0.030, 0.26]Fiber (% DW)Acid Detergent Fiber16.35 (0.29)16.74 (0.29)-0.38 (0.29)-1.61, 0.850.313(12.46 - 21.25)[16.22 - 16.63][16.03 - 17.32][-0.69 - 0.19][12.71, 19.29]Neutral Detergent Fiber20.24 (0.63)17.83 (0.63)2.42 (0.89)-1.40, 6.230.112(12.25 - 20.89)[18.92 - 21.65][17.32 - 18.62][0.30 - 4.33][12.07, 21.51]Proximate (% DW)Ash6.11 (0.11)6.41 (0.11)-0.30 (0.14)-0.90, 0.300.164(5.64 - 6.82)[5.99 - 6.26][6.14 - 6.55][-0.56 - -0.073][5.26, 7.17]Monsanto Company 09-SY-201UPage 259 of 471Table E-4 (continued). Statistical Summaryof Site MEL Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Proximate (% DW)Carbohydrates39.70 (0.55)39.60 (0.55)0.10 (0.59)-2.43, 2.640.876(32.79 - 42.29)[38.85 - 41.07][38.91 - 40.04][-0.99 - 1.03][30.78, 45.86]Moisture (% FW)10.93 (0.21)11.37 (0.21)-0.43 (0.15)-1.06, 0.190.096(6.89 - 12.50)[10.40 - 11.20][11.10 - 11.60][-0.70 - -0.20][5.51, 13.37]Protein35.56 (0.92)34.38 (0.92)1.18 (0.95)-2.89, 5.260.337(29.51 - 40.25)[33.59 - 36.94][33.30 - 35.97][0.18 - 3.08][26.12, 43.51]Total Fat18.64 (0.29)19.63 (0.29)-0.99 (0.31)-2.35, 0.360.087(16.91 - 23.48)[18.24 - 19.08][19.00 - 20.13][-1.51 - -0.42][15.35, 25.95]Vitamin (mg/100g DW)Vitamin E3.26 (0.11)3.83 (0.11)-0.58 (0.043)-0.76, -0.390.005(1.09 - 5.10)[3.15 - 3.45][3.66 - 4.05][-0.64 - -0.49][0, 7.36]AntinutrientLectin (H.U./mg DW)1.84 (0.30)1.69 (0.30)0.15 (0.42)-1.68, 1.980.757(0.65 - 8.10)[1.61 - 2.14][1.04 - 2.40][-0.63 - 1.11][0, 6.44]Monsanto Company 09-SY-201UPage 260 of 471Table E-4 (continued). Statistical Summaryof Site MEL Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Antinutrient (% DW)Phytic Acid1.89 (0.099)1.90 (0.099)-0.0094 (0.14)-0.60, 0.580.951(1.42 - 2.27)[1.80 - 2.02][1.66 - 2.08][-0.27 - 0.18][1.35, 2.35]Raffinose0.57 (0.019)0.56 (0.019)0.018 (0.0085)-0.019, 0.0540.175(0.40 - 0.80)[0.54 - 0.61][0.53 - 0.58][0.0046 - 0.034][0.27, 0.87]Stachyose3.68 (0.13)3.78 (0.13)-0.10 (0.18)-0.87, 0.660.615(2.30 - 4.53)[3.56 - 3.77][3.51 - 4.08][-0.53 - 0.26][1.96, 4.41]Trypsin Inhibitor (TIU/mg DW)35.12 (2.90)36.06 (2.90)-0.93 (1.36)-6.80, 4.930.564(23.11 - 60.42)[31.64 - 41.07][31.67 - 41.28][-3.57 - 0.98][8.75, 63.43]Isoflavone (μg/g DW)Daidzein2095.83 (90.09)2012.16 (90.09)83.67 (127.41)-464.54, 631.880.578(320.54 - 3061.22)[2060.81 - 2139.64][1844.77 - 2257.34][-196.53 - 242.28][0, 3328.03]Genistein1362.28 (58.14)1286.17 (58.14)76.11 (82.22)-277.67, 429.880.452(433.41 - 2301.59)[1340.09 - 1385.14][1176.47 - 1444.70][-104.61 - 208.66][0, 2727.33]Monsanto Company 09-SY-201UPage 261 of 471Table E-4 (continued). Statistical Summaryof Site MEL Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Glycitein144.06 (9.70)142.17 (9.70)1.90 (13.72)-57.13, 60.930.902(21.67 - 354.30)[132.88 - 151.79][128.23 - 167.04][-34.16 - 23.55][0, 376.03]1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standarderror; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 262 of 471Table E-5. Statistical Summary of Site QUI Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Acid Detergent Fiber24.62 (1.87)22.74 (2.29)1.88 (2.96)-35.75, 39.510.639(23.18 - 42.11)[20.04 - 26.98][22.72 - 22.76][4.10 - 4.25][18.29, 41.02]Ash8.26 (0.32)7.87 (0.39)0.39 (0.50)-5.97, 6.760.575(6.76 - 10.40)[8.02 - 8.51][7.24 - 8.49][-0.24 - 1.27][6.78, 9.91]Carbohydrates71.07 (0.43)72.76 (0.53)-1.69 (0.68)-10.36, 6.980.244(63.74 - 80.60)[70.11 - 71.67][72.40 - 73.12][-3.01 - -0.73][64.45, 80.50]Moisture (% FW)71.77 (0.47)72.10 (0.58)-0.33 (0.75)-9.87, 9.200.733(65.80 - 82.00)[70.70 - 72.70][72.10 - 72.10][-1.40 - -0.20][62.26, 83.45]Neutral Detergent Fiber30.94 (2.43)32.99 (2.51)-2.05 (1.09)-15.95, 11.850.312(24.70 - 46.55)[26.12 - 33.59][33.91 - 36.56][-2.96 - -0.80][22.57, 46.52]Protein16.36 (0.79)15.09 (0.97)1.27 (1.26)-14.70, 17.240.496(9.51 - 19.93)[15.22 - 18.10][14.37 - 15.81][-0.58 - 1.39][7.38, 21.27]Monsanto Company 09-SY-201UPage 263 of 471Table E-5 (continued). Statistical Summary of Site QUI Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Total Fat4.35 (1.10)3.42 (1.15)0.93 (0.57)-6.30, 8.160.349(1.19 - 8.22)[2.29 - 5.80][3.36 - 5.34][0.46 - 1.59][0, 9.74]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 264 of 471Table E-6. Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid,Fiber, Proximate, Vitamin, Antinutrientand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.64 (0.031)1.68 (0.031)-0.036 (0.0029)-0.072, 0.000380.050(1.34 - 1.78)[1.60 - 1.70][1.63 - 1.66][-0.039 - -0.033][1.25, 1.92]Arginine2.96 (0.090)2.98 (0.091)-0.022 (0.028)-0.38, 0.330.577(2.15 - 3.23)[2.82 - 3.12][2.82 - 2.99][-0.052 - 0.0034][1.81, 3.62]Aspartic Acid4.31 (0.10)4.40 (0.11)-0.095 (0.053)-0.77, 0.580.323(3.37 - 4.76)[4.16 - 4.49][4.21 - 4.43][-0.16 - -0.051][3.02, 5.11]Cystine0.63 (0.0098)0.62 (0.011)0.0059 (0.010)-0.12, 0.140.666(0.53 - 0.64)[0.60 - 0.64][0.61 - 0.63][-0.0061 - 0.017][0.49, 0.69]Glutamic Acid6.85 (0.20)7.06 (0.20)-0.21 (0.058)-0.94, 0.520.171(5.14 - 7.73)[6.54 - 7.19][6.70 - 7.09][-0.27 - -0.16][4.42, 8.48]Glycine1.64 (0.029)1.65 (0.035)-0.012 (0.046)-0.59, 0.570.837(1.30 - 1.79)[1.60 - 1.71][1.65 - 1.65][-0.050 - -0.045][1.19, 1.95]Monsanto Company 09-SY-201UPage 265 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Histidine0.99 (0.024)1.01 (0.024)-0.018 (0.0035)-0.062, 0.0270.124(0.79 - 1.07)[0.95 - 1.04][0.97 - 1.01][-0.021 - -0.014][0.74, 1.16]Isoleucine1.76 (0.054)1.87 (0.058)-0.10 (0.036)-0.56, 0.360.214(1.37 - 2.00)[1.65 - 1.86][1.80 - 1.86][-0.15 - -0.074][1.23, 2.15]Leucine2.88 (0.073)2.94 (0.074)-0.061 (0.022)-0.34, 0.220.220(2.26 - 3.14)[2.77 - 3.01][2.81 - 2.94][-0.085 - -0.041][2.06, 3.41]Lysine2.48 (0.043)2.50 (0.044)-0.019 (0.016)-0.22, 0.180.438(2.00 - 2.63)[2.41 - 2.55][2.42 - 2.51][-0.036 - -0.0050][1.87, 2.81]Methionine0.55 (0.0071)0.56 (0.0087)-0.0063 (0.011)-0.15, 0.140.677(0.46 - 0.59)[0.54 - 0.57][0.55 - 0.57][-0.022 - -0.0066][0.43, 0.63]Phenylalanine1.89 (0.053)1.93 (0.065)-0.044 (0.084)-1.11, 1.030.695(1.50 - 2.11)[1.83 - 2.00][1.88 - 1.99][-0.16 - -0.035][1.35, 2.31]Monsanto Company 09-SY-201UPage 266 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Proline1.85 (0.045)1.88 (0.045)-0.032 (0.0091)-0.15, 0.0830.176(1.43 - 2.03)[1.77 - 1.92][1.80 - 1.90][-0.041 - -0.023][1.29, 2.21]Serine1.92 (0.051)1.83 (0.062)0.083 (0.080)-0.93, 1.100.486(1.55 - 2.05)[1.87 - 1.98][1.74 - 1.92][-0.053 - 0.16][1.44, 2.15]Threonine1.42 (0.023)1.37 (0.028)0.046 (0.037)-0.42, 0.510.429(1.19 - 1.48)[1.38 - 1.45][1.34 - 1.41][-0.025 - 0.11][1.12, 1.53]Tryptophan0.43 (0.0092)0.44 (0.011)-0.0041 (0.015)-0.19, 0.180.826(0.33 - 0.48)[0.41 - 0.45][0.43 - 0.44][-0.025 - -0.0021][0.30, 0.50]Tyrosine1.31 (0.018)1.28 (0.021)0.024 (0.028)-0.33, 0.380.543(1.07 - 1.39)[1.29 - 1.32][1.25 - 1.32][-0.024 - 0.059][0.99, 1.49]Valine1.86 (0.059)1.96 (0.066)-0.10 (0.053)-0.77, 0.560.300(1.45 - 2.13)[1.73 - 1.96][1.90 - 1.96][-0.17 - -0.064][1.31, 2.29]Monsanto Company 09-SY-201UPage 267 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)16:0 Palmitic2.30 (0.042)10.56 (0.051)-8.26 (0.066)-9.10, -7.430.005(8.78 - 11.51)[2.25 - 2.37][10.51 - 10.62][-8.37 - -8.13][7.62, 12.55]18:0 Stearic3.51 (0.19)4.82 (0.19)-1.31 (0.0088)-1.42, -1.200.004(3.82 - 7.21)[3.15 - 3.82][4.47 - 4.85][-1.32 - -1.30][2.87, 7.15]18:1 Oleic78.61 (0.41)24.95 (0.44)53.66 (0.26)50.35, 56.960.003(20.77 - 27.19)[77.70 - 79.17][24.34 - 25.08][53.36 - 53.88][18.40, 30.22]18:2 Linoleic8.75 (0.58)51.70 (0.63)-42.95 (0.44)-48.50, -37.390.006(48.62 - 54.74)[7.85 - 10.02][51.68 - 52.44][-43.30 - -42.42][47.75, 56.46]18:3 Linolenic5.64 (0.074)7.02 (0.082)-1.38 (0.063)-2.19, -0.580.029(5.89 - 9.11)[5.55 - 5.71][6.86 - 7.16][-1.45 - -1.32][4.97, 9.93]20:0 Arachidic0.33 (0.017)0.36 (0.017)-0.032 (0.0021)-0.058, -0.00550.041(0.28 - 0.54)[0.30 - 0.36][0.33 - 0.36][-0.034 - -0.030][0.22, 0.53]Monsanto Company 09-SY-201UPage 268 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic0.38 (0.0089)0.20 (0.011)0.18 (0.014)0.0019, 0.360.049(0.15 - 0.22)[0.37 - 0.40][0.20 - 0.20][0.18 - 0.20][0.13, 0.25]22:0 Behenic0.31 (0.0094)0.31 (0.0095)0.0051 (0.0029)-0.032, 0.0420.330(0.29 - 0.46)[0.30 - 0.33][0.29 - 0.31][0.0019 - 0.0078][0.22, 0.47]24:0 Lignoceric0.17 (0.0027)0.15 (0.0033)0.021 (0.0042)-0.032, 0.0750.124(0.056 - 0.21)[0.17 - 0.17][0.15 - 0.16][0.015 - 0.027][0.030, 0.26]Fiber (% DW)Acid Detergent Fiber18.15 (0.59)15.96 (0.73)2.19 (0.94)-9.75, 14.130.258(12.46 - 21.25)[17.23 - 19.31][15.28 - 16.65][1.28 - 4.03][12.71, 19.29]Neutral Detergent Fiber18.01 (0.71)16.53 (0.87)1.48 (1.13)-12.86, 15.820.414(12.25 - 20.89)[16.93 - 19.69][16.18 - 16.87][0.054 - 1.23][12.07, 21.51]Proximate (% DW)Ash6.22 (0.089)6.10 (0.10)0.12 (0.091)-1.04, 1.280.414(5.64 - 6.82)[6.06 - 6.42][6.13 - 6.18][0.061 - 0.24][5.26, 7.17]Monsanto Company 09-SY-201UPage 269 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Proximate (% DW)Carbohydrates37.54 (0.51)37.41 (0.55)0.12 (0.34)-4.20, 4.440.779(32.79 - 42.29)[36.69 - 38.62][37.46 - 38.09][-0.15 - 0.53][30.78, 45.86]Moisture (% FW)10.40 (0.097)11.05 (0.12)-0.65 (0.15)-2.60, 1.300.147(6.89 - 12.50)[10.20 - 10.60][11.00 - 11.10][-0.90 - -0.60][5.51, 13.37]Protein37.05 (0.83)37.11 (0.84)-0.055 (0.27)-3.50, 3.390.873(29.51 - 40.25)[35.60 - 38.59][35.96 - 36.78][-0.35 - 0.19][26.12, 43.51]Total Fat19.20 (0.21)19.73 (0.26)-0.53 (0.34)-4.80, 3.730.356(16.91 - 23.48)[18.68 - 19.49][19.69 - 19.78][-0.36 - -0.20][15.35, 25.95]Vitamin (mg/100g DW)Vitamin E3.93 (0.19)3.72 (0.23)0.21 (0.29)-3.51, 3.930.599(1.09 - 5.10)[3.70 - 4.36][3.61 - 3.82][-0.13 - 0.76][0, 7.36]AntinutrientLectin (H.U./mg DW)1.45 (0.82)3.92 (1.00)-2.47 (1.30)-18.93, 13.990.307(0.65 - 8.10)[0.72 - 2.03][2.32 - 5.53][-4.80 - -0.29][0, 6.44]Monsanto Company 09-SY-201UPage 270 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Antinutrient (% DW)Phytic Acid1.89 (0.12)1.97 (0.12)-0.085 (0.024)-0.39, 0.220.178(1.42 - 2.27)[1.73 - 2.13][1.90 - 2.19][-0.11 - -0.059][1.35, 2.35]Raffinose0.60 (0.023)0.55 (0.029)0.049 (0.037)-0.42, 0.520.412(0.40 - 0.80)[0.56 - 0.64][0.52 - 0.57][-0.015 - 0.12][0.27, 0.87]Stachyose4.08 (0.31)3.84 (0.38)0.24 (0.49)-5.93, 6.420.703(2.30 - 4.53)[3.43 - 4.48][3.52 - 4.16][0.18 - 0.96][1.96, 4.41]Trypsin Inhibitor (TIU/mg DW)42.52 (5.28)40.77 (5.31)1.75 (0.99)-10.88, 14.380.329(23.11 - 60.42)[33.22 - 52.01][41.51 - 49.21][0.81 - 2.80][8.75, 63.43]Isoflavone (μg/g DW)Daidzein1346.63 (67.43)1411.91 (69.07)-65.28 (26.14)-397.44, 266.880.242(320.54 - 3061.22)[1230.43 - 1450.89][1394.83 - 1539.33][-88.43 - -36.25][0, 3328.03]Genistein952.69 (44.70)932.29 (52.06)20.40 (50.70)-623.77, 664.560.756(433.41 - 2301.59)[882.55 - 1005.58][881.89 - 1020.22][-14.64 - 88.04][0, 2727.33]Monsanto Company 09-SY-201UPage 271 of 471Table E-6 (continued). Statistical Summaryof Site QUI Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Glycitein87.69 (17.12)98.58 (18.14)-10.89 (10.64)-146.05, 124.280.492(21.67 - 354.30)[49.11 - 108.04][72.10 - 109.21][-22.99 - -1.18][0, 376.03]1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standarderror; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 272 of 471Table E-7. Statistical Summary of Site RAN Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Acid Detergent Fiber32.11 (2.53)29.37 (2.53)2.73 (3.57)-12.65, 18.110.524(23.18 - 42.11)[31.02 - 33.39][24.37 - 36.13][-4.22 - 9.02][18.29, 41.02]Ash9.64 (0.30)8.56 (0.30)1.07 (0.42)-0.75, 2.900.126(6.76 - 10.40)[8.81 - 10.11][8.40 - 8.67][0.14 - 1.60][6.78, 9.91]Carbohydrates69.77 (0.62)72.09 (0.62)-2.32 (0.40)-4.02, -0.620.027(63.74 - 80.60)[68.94 - 71.06][70.97 - 72.89][-3.10 - -1.83][64.45, 80.50]Moisture (% FW)78.03 (1.32)75.53 (1.32)2.50 (1.86)-5.50, 10.500.311(65.80 - 82.00)[76.50 - 81.10][73.90 - 77.50][-1.00 - 7.20][62.26, 83.45]Neutral Detergent Fiber41.60 (1.83)36.03 (1.83)5.58 (1.75)-1.97, 13.120.086(24.70 - 46.55)[39.19 - 46.30][33.91 - 37.55][2.70 - 8.75][22.57, 46.52]Protein16.73 (0.63)14.71 (0.63)2.02 (0.89)-1.82, 5.870.151(9.51 - 19.93)[15.19 - 17.78][14.02 - 15.48][0.57 - 3.75][7.38, 21.27]Monsanto Company 09-SY-201UPage 273 of 471Table E-7 (continued). Statistical Summary of Site RAN Soybean Forage Fiber and Proximate Content for MON 87705 vs.the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Total Fat3.87 (0.48)4.66 (0.48)-0.80 (0.67)-3.69, 2.100.358(1.19 - 8.22)[2.83 - 4.89][4.05 - 5.06][-2.23 - 0.015][0, 9.74]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 274 of 471Table E-8. Statistical Summary of Site RANSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitamin, Antinutrientand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.66 (0.021)1.62 (0.021)0.036 (0.027)-0.080, 0.150.314(1.34 - 1.78)[1.62 - 1.70][1.59 - 1.66][-0.0069 - 0.085][1.25, 1.92]Arginine3.08 (0.032)2.95 (0.032)0.13 (0.045)-0.062, 0.330.099(2.15 - 3.23)[3.01 - 3.16][2.94 - 2.97][0.043 - 0.22][1.81, 3.62]Aspartic Acid4.35 (0.036)4.24 (0.036)0.11 (0.051)-0.11, 0.330.173(3.37 - 4.76)[4.30 - 4.44][4.20 - 4.29][0.026 - 0.23][3.02, 5.11]Cystine0.60 (0.011)0.58 (0.011)0.022 (0.011)-0.024, 0.0690.171(0.53 - 0.64)[0.57 - 0.62][0.57 - 0.59][0.00097 - 0.033][0.49, 0.69]Glutamic Acid7.00 (0.060)6.82 (0.060)0.17 (0.085)-0.19, 0.540.177(5.14 - 7.73)[6.92 - 7.14][6.73 - 6.89][0.039 - 0.41][4.42, 8.48]Glycine1.68 (0.030)1.63 (0.030)0.046 (0.020)-0.041, 0.130.150(1.30 - 1.79)[1.62 - 1.74][1.58 - 1.67][0.015 - 0.084][1.19, 1.95]Monsanto Company 09-SY-201UPage 275 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Histidine1.02 (0.0088)0.99 (0.0088)0.031 (0.012)-0.022, 0.0850.127(0.79 - 1.07)[1.01 - 1.04][0.98 - 1.01][0.0086 - 0.060][0.74, 1.16]Isoleucine1.83 (0.022)1.76 (0.022)0.069 (0.031)-0.063, 0.200.153(1.37 - 2.00)[1.78 - 1.88][1.74 - 1.77][0.0045 - 0.14][1.23, 2.15]Leucine2.91 (0.022)2.86 (0.022)0.049 (0.031)-0.085, 0.180.256(2.26 - 3.14)[2.89 - 2.97][2.85 - 2.90][-0.0094 - 0.12][2.06, 3.41]Lysine2.49 (0.021)2.43 (0.021)0.065 (0.030)-0.065, 0.190.164(2.00 - 2.63)[2.46 - 2.55][2.41 - 2.45][0.018 - 0.13][1.87, 2.81]Methionine0.57 (0.0092)0.53 (0.0092)0.035 (0.010)-0.0098, 0.0790.078(0.46 - 0.59)[0.55 - 0.58][0.52 - 0.54][0.015 - 0.049][0.43, 0.63]Phenylalanine1.95 (0.013)1.92 (0.013)0.028 (0.018)-0.050, 0.110.265(1.50 - 2.11)[1.93 - 1.98][1.90 - 1.94][-0.0066 - 0.075][1.35, 2.31]Monsanto Company 09-SY-201UPage 276 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Proline1.92 (0.018)1.83 (0.018)0.093 (0.025)-0.014, 0.200.064(1.43 - 2.03)[1.88 - 1.95][1.80 - 1.85][0.026 - 0.14][1.29, 2.21]Serine1.79 (0.041)1.91 (0.041)-0.11 (0.058)-0.37, 0.140.186(1.55 - 2.05)[1.74 - 1.90][1.88 - 1.94][-0.20 - 0.0033][1.44, 2.15]Threonine1.36 (0.014)1.36 (0.014)-0.00098 (0.020)-0.088, 0.0860.965(1.19 - 1.48)[1.33 - 1.39][1.34 - 1.38][-0.052 - 0.042][1.12, 1.53]Tryptophan0.45 (0.0040)0.44 (0.0040)0.0065 (0.0057)-0.018, 0.0310.372(0.33 - 0.48)[0.44 - 0.46][0.44 - 0.44][-0.0022 - 0.022][0.30, 0.50]Tyrosine1.29 (0.014)1.30 (0.014)-0.0067 (0.014)-0.065, 0.0520.670(1.07 - 1.39)[1.26 - 1.33][1.29 - 1.31][-0.026 - 0.019][0.99, 1.49]Valine1.97 (0.023)1.89 (0.023)0.080 (0.033)-0.063, 0.220.137(1.45 - 2.13)[1.91 - 2.02][1.87 - 1.91][0.026 - 0.15][1.31, 2.29]Monsanto Company 09-SY-201UPage 277 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)16:0 Palmitic2.40 (0.0088)10.96 (0.0088)-8.56 (0.012)-8.61, -8.51<0.001(8.78 - 11.51)[2.39 - 2.40][10.94 - 10.98][-8.58 - -8.54][7.62, 12.55]18:0 Stearic3.34 (0.032)4.50 (0.032)-1.16 (0.045)-1.35, -0.960.001(3.82 - 7.21)[3.28 - 3.41][4.47 - 4.55][-1.22 - -1.07][2.87, 7.15]18:1 Oleic74.69 (0.59)21.53 (0.59)53.15 (0.79)49.76, 56.55<0.001(20.77 - 27.19)[73.13 - 75.98][21.41 - 21.63][51.71 - 54.43][18.40, 30.22]18:2 Linoleic11.32 (0.42)53.73 (0.42)-42.41 (0.57)-44.84, -39.97<0.001(48.62 - 54.74)[10.37 - 12.42][53.67 - 53.81][-43.33 - -41.38][47.75, 56.46]18:3 Linolenic7.32 (0.19)8.43 (0.19)-1.11 (0.27)-2.28, 0.0660.055(5.89 - 9.11)[6.87 - 7.81][8.40 - 8.47][-1.60 - -0.59][4.97, 9.93]20:0 Arachidic0.28 (0.0036)0.33 (0.0036)-0.043 (0.0051)-0.065, -0.0210.014(0.28 - 0.54)[0.28 - 0.29][0.32 - 0.33][-0.053 - -0.031][0.22, 0.53]Monsanto Company 09-SY-201UPage 278 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic0.29 (0.0087)0.16 (0.0087)0.13 (0.0081)0.096, 0.170.003(0.15 - 0.22)[0.27 - 0.31][0.15 - 0.16][0.12 - 0.15][0.13, 0.25]22:0 Behenic0.28 (0.0041)0.30 (0.0041)-0.016 (0.0047)-0.036, 0.00480.080(0.29 - 0.46)[0.28 - 0.29][0.30 - 0.30][-0.021 - -0.0062][0.22, 0.47]24:0 Lignoceric0.069 (0.0011)0.067 (0.0011)0.0017 (0.0012)-0.0034, 0.00690.285(0.056 - 0.21)[0.066 - 0.071][0.067 - 0.068][-0.00065 -0.0032][0.030, 0.26]Fiber(% DW)Acid Detergent Fiber16.32 (0.34)13.94 (0.34)2.38 (0.11)1.91, 2.850.002(12.46 - 21.25)[15.71 - 16.78][13.36 - 14.58][2.20 - 2.58][12.71, 19.29]Neutral Detergent Fiber15.14 (0.91)16.40 (0.91)-1.26 (0.39)-2.95, 0.430.085(12.25 - 20.89)[13.41 - 16.48][14.61 - 17.51][-1.97 - -0.61][12.07, 21.51]Proximate (% DW)Ash5.53 (0.088)5.68 (0.088)-0.14 (0.12)-0.68, 0.390.370(5.64 - 6.82)[5.46 - 5.63][5.48 - 5.88][-0.42 - 0.15][5.26, 7.17]Monsanto Company 09-SY-201UPage 279 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Proximate (% DW)Carbohydrates40.12 (0.24)39.60 (0.24)0.52 (0.34)-0.93, 1.970.262(32.79 - 42.29)[39.56 - 40.64][39.41 - 39.84][-0.28 - 1.23][30.78, 45.86]Moisture (% FW)9.52 (0.43)12.03 (0.43)-2.51 (0.61)-5.12, 0.0900.053(6.89 - 12.50)[8.96 - 10.60][11.50 - 12.40][-3.40 - -0.90][5.51, 13.37]Protein37.54 (0.20)36.87 (0.20)0.67 (0.19)-0.16, 1.510.074(29.51 - 40.25)[37.35 - 37.70][36.56 - 37.40][0.29 - 0.94][26.12, 43.51]Total Fat16.83 (0.21)17.85 (0.21)-1.01 (0.30)-2.30, 0.270.077(16.91 - 23.48)[16.55 - 17.36][17.63 - 18.11][-1.52 - -0.45][15.35, 25.95]Vitamin (mg/100g DW)Vitamin E1.44 (0.094)1.81 (0.094)-0.38 (0.094)-0.78, 0.0260.056(1.09 - 5.10)[1.23 - 1.64][1.69 - 1.91][-0.47 - -0.19][0, 7.36]AntinutrientLectin (H.U./mg DW)3.57 (0.50)1.36 (0.50)2.21 (0.61)-0.39, 4.820.067(0.65 - 8.10)[3.30 - 3.77][0.61 - 2.73][1.04 - 3.04][0, 6.44]Monsanto Company 09-SY-201UPage 280 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Antinutrient (% DW)Phytic Acid1.62 (0.032)1.72 (0.032)-0.10 (0.042)-0.28, 0.0790.139(1.42 - 2.27)[1.61 - 1.63][1.63 - 1.77][-0.14 - -0.016][1.35, 2.35]Raffinose0.52 (0.017)0.56 (0.017)-0.044 (0.023)-0.14, 0.0560.198(0.40 - 0.80)[0.48 - 0.56][0.55 - 0.57][-0.088 - -0.011][0.27, 0.87]Stachyose3.79 (0.21)3.70 (0.21)0.084 (0.30)-1.20, 1.370.804(2.30 - 4.53)[3.39 - 4.35][3.56 - 3.80][-0.35 - 0.79][1.96, 4.41]Trypsin Inhibitor (TIU/mg DW)29.38 (1.10)28.54 (1.10)0.85 (0.95)-3.23, 4.920.465(23.11 - 60.42)[26.73 - 31.10][27.23 - 29.95][-0.50 - 2.67][8.75, 63.43]Isoflavone (μg/g DW)Daidzein1915.89 (53.73)1947.43 (53.73)-31.54 (48.74)-241.26, 178.180.583(320.54 - 3061.22)[1868.13 - 1946.31][1803.65 - 2027.33][-94.12 - 64.48][0, 3328.03]Genistein1186.26 (34.98)1149.45 (34.98)36.81 (39.06)-131.24, 204.870.445(433.41 - 2301.59)[1164.84 - 1208.26][1054.79 - 1209.04][-23.36 - 110.04][0, 2727.33]Monsanto Company 09-SY-201UPage 281 of 471Table E-8 (continued). Statistical Summaryof Site RAN Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Glycitein117.22 (15.47)130.37 (15.47)-13.15 (21.88)-107.29, 80.990.608(21.67 - 354.30)[79.19 - 140.66][110.84 - 146.92][-54.14 - 29.81][0, 376.03]1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standarderror; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 282 of 471Table E-9. Statistical Summary of Site SFR Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Acid Detergent Fiber34.33 (3.08)38.52 (3.08)-4.19 (4.36)-22.93, 14.550.437(23.18 - 42.11)[30.23 - 42.14][34.68 - 40.67][-10.04 - 7.47][18.29, 41.02]Ash8.54 (0.51)7.73 (0.51)0.81 (0.73)-2.31, 3.940.379(6.76 - 10.40)[7.39 - 9.74][7.21 - 8.02][-0.63 - 1.79][6.78, 9.91]Carbohydrates76.37 (1.00)78.35 (1.00)-1.98 (1.42)-8.09, 4.130.297(63.74 - 80.60)[74.24 - 78.93][77.70 - 78.99][-4.74 - 1.23][64.45, 80.50]Moisture (% FW)72.87 (0.39)72.60 (0.39)0.27 (0.47)-1.74, 2.270.625(65.80 - 82.00)[72.00 - 73.60][72.20 - 73.20][-0.20 - 1.20][62.26, 83.45]Neutral Detergent Fiber38.24 (1.13)43.34 (1.13)-5.10 (1.53)-11.67, 1.480.079(24.70 - 46.55)[37.27 - 38.89][40.65 - 45.90][-7.01 - -2.08][22.57, 46.52]Protein9.84 (0.24)9.77 (0.24)0.070 (0.32)-1.29, 1.430.845(9.51 - 19.93)[9.25 - 10.42][9.71 - 9.81][-0.46 - 0.63][7.38, 21.27]Monsanto Company 09-SY-201UPage 283 of 471Table E-9 (continued). Statistical Summaryof Site SFR Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Total Fat5.25 (0.64)4.31 (0.64)0.93 (0.91)-2.98, 4.840.413(1.19 - 8.22)[4.36 - 7.01][3.99 - 4.75][-0.39 - 2.80][0, 9.74]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 284 of 471Table E-10. Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitamin, Antinutrientand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.51 (0.011)1.44 (0.011)0.067 (0.011)0.021, 0.110.024(1.34 - 1.78)[1.49 - 1.54][1.43 - 1.45][0.055 - 0.088][1.25, 1.92]Arginine2.52 (0.048)2.34 (0.048)0.18 (0.040)0.0051, 0.350.047(2.15 - 3.23)[2.43 - 2.64][2.31 - 2.39][0.11 - 0.24][1.81, 3.62]Aspartic Acid3.76 (0.054)3.56 (0.054)0.20 (0.019)0.11, 0.280.009(3.37 - 4.76)[3.67 - 3.88][3.51 - 3.65][0.17 - 0.23][3.02, 5.11]Cystine0.61 (0.010)0.57 (0.010)0.043 (0.015)-0.020, 0.110.098(0.53 - 0.64)[0.60 - 0.62][0.55 - 0.59][0.0062 - 0.073][0.49, 0.69]Glutamic Acid5.90 (0.11)5.53 (0.11)0.37 (0.034)0.22, 0.510.008(5.14 - 7.73)[5.72 - 6.12][5.42 - 5.71][0.30 - 0.41][4.42, 8.48]Glycine1.49 (0.015)1.42 (0.015)0.078 (0.022)-0.016, 0.170.069(1.30 - 1.79)[1.47 - 1.54][1.41 - 1.43][0.055 - 0.12][1.19, 1.95]Monsanto Company 09-SY-201UPage 285 of 471Table-10 (continued). StatisticalSummary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber,Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Histidine0.90 (0.014)0.85 (0.014)0.050 (0.0069)0.021, 0.0800.018(0.79 - 1.07)[0.88 - 0.94][0.84 - 0.87][0.043 - 0.064][0.74, 1.16]Isoleucine1.60 (0.029)1.51 (0.029)0.093 (0.030)-0.037, 0.220.091(1.37 - 2.00)[1.56 - 1.66][1.45 - 1.54][0.032 - 0.12][1.23, 2.15]Leucine2.54 (0.036)2.41 (0.036)0.12 (0.015)0.059, 0.190.014(2.26 - 3.14)[2.47 - 2.61][2.37 - 2.47][0.095 - 0.15][2.06, 3.41]Lysine2.25 (0.026)2.13 (0.026)0.11 (0.010)0.070, 0.160.007(2.00 - 2.63)[2.19 - 2.30][2.10 - 2.17][0.097 - 0.13][1.87, 2.81]Methionine0.53 (0.0080)0.50 (0.0080)0.037 (0.011)-0.011, 0.0860.081(0.46 - 0.59)[0.52 - 0.55][0.49 - 0.51][0.0074 - 0.056][0.43, 0.63]Phenylalanine1.68 (0.023)1.60 (0.023)0.077 (0.011)0.030, 0.120.019(1.50 - 2.11)[1.64 - 1.73][1.58 - 1.64][0.056 - 0.089][1.35, 2.31]Monsanto Company 09-SY-201UPage 286 of 471Table E-10 (continued). Statistical Summaryof Site SFR Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Proline1.62 (0.023)1.55 (0.023)0.078 (0.012)0.028, 0.130.021(1.43 - 2.03)[1.59 - 1.66][1.52 - 1.60][0.065 - 0.10][1.29, 2.21]Serine1.56 (0.063)1.58 (0.063)-0.017 (0.089)-0.40, 0.360.863(1.55 - 2.05)[1.49 - 1.68][1.45 - 1.68][-0.15 - 0.23][1.44, 2.15]Threonine1.24 (0.022)1.22 (0.022)0.022 (0.032)-0.11, 0.160.559(1.19 - 1.48)[1.20 - 1.28][1.18 - 1.25][-0.044 - 0.10][1.12, 1.53]Tryptophan0.39 (0.011)0.37 (0.011)0.024 (0.016)-0.045, 0.0920.276(0.33 - 0.48)[0.37 - 0.41][0.35 - 0.38][-0.0073 - 0.046][0.30, 0.50]Tyrosine1.18 (0.0097)1.12 (0.0097)0.064 (0.014)0.0052, 0.120.042(1.07 - 1.39)[1.17 - 1.20][1.10 - 1.13][0.039 - 0.10][0.99, 1.49]Valine1.72 (0.031)1.61 (0.031)0.12 (0.036)-0.039, 0.270.084(1.45 - 2.13)[1.69 - 1.79][1.55 - 1.64][0.044 - 0.16][1.31, 2.29]Monsanto Company 09-SY-201UPage 287 of 471Table E-10 (continued). Statistical Summaryof Site SFR Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)16:0 Palmitic2.42 (0.029)11.00 (0.029)-8.58 (0.042)-8.76, -8.40<0.001(8.78 - 11.51)[2.40 - 2.44][10.95 - 11.08][-8.68 - -8.53][7.62, 12.55]18:0 Stearic3.22 (0.084)4.31 (0.084)-1.09 (0.044)-1.28, -0.900.001(3.82 - 7.21)[3.07 - 3.41][4.24 - 4.44][-1.17 - -1.03][2.87, 7.15]18:1 Oleic76.49 (0.43)22.42 (0.43)54.07 (0.60)51.47, 56.67<0.001(20.77 - 27.19)[75.33 - 77.21][22.16 - 22.65][52.87 - 55.05][18.40, 30.22]18:2 Linoleic9.82 (0.27)52.84 (0.27)-43.02 (0.38)-44.64, -41.39<0.001(48.62 - 54.74)[9.33 - 10.55][52.75 - 52.98][-43.65 - -42.21][47.75, 56.46]18:3 Linolenic6.98 (0.11)8.49 (0.11)-1.50 (0.15)-2.16, -0.850.009(5.89 - 9.11)[6.79 - 7.26][8.41 - 8.60][-1.80 - -1.20][4.97, 9.93]20:0 Arachidic0.29 (0.0035)0.32 (0.0035)-0.026 (0.0021)-0.035, -0.0170.006(0.28 - 0.54)[0.29 - 0.29][0.31 - 0.32][-0.029 - -0.022][0.22, 0.53]Monsanto Company 09-SY-201UPage 288 of 471Table E-10 (continued). Statistical Summaryof Site SFR Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic0.33 (0.0077)0.18 (0.0077)0.15 (0.011)0.10, 0.190.005(0.15 - 0.22)[0.32 - 0.35][0.17 - 0.19][0.13 - 0.17][0.13, 0.25]22:0 Behenic0.29 (0.0025)0.30 (0.0025)-0.0071 (0.0033)-0.021, 0.00690.161(0.29 - 0.46)[0.29 - 0.29][0.29 - 0.30][-0.012 - -0.0012][0.22, 0.47]24:0 Lignoceric0.15 (0.0045)0.14 (0.0045)0.0090 (0.0039)-0.0077, 0.0260.145(0.056 - 0.21)[0.14 - 0.16][0.13 - 0.14][0.0013 - 0.014][0.030, 0.26]Fiber (% DW)Acid Detergent Fiber16.62 (0.28)17.78 (0.28)-1.17 (0.36)-2.74, 0.400.085(12.46 - 21.25)[15.85 - 17.05][17.64 - 18.02][-1.84 - -0.59][12.71, 19.29]Neutral Detergent Fiber17.75 (0.93)19.99 (0.93)-2.24 (1.01)-6.57, 2.090.156(12.25 - 20.89)[15.62 - 19.68][19.01 - 21.09][-4.24 - -1.06][12.07, 21.51]Proximate (% DW)Ash6.05 (0.10)6.17 (0.10)-0.12 (0.14)-0.73, 0.490.491(5.64 - 6.82)[5.84 - 6.32][6.15 - 6.22][-0.38 - 0.16][5.26, 7.17]Monsanto Company 09-SY-201UPage 289 of 471Table E-10 (continued). Statistical Summaryof Site SFR Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Proximate (% DW)Carbohydrates42.56 (0.37)42.80 (0.37)-0.24 (0.47)-2.28, 1.800.663(32.79 - 42.29)[41.73 - 43.52][42.65 - 42.96][-1.08 - 0.57][30.78, 45.86]Moisture (% FW)12.13 (0.11)12.47 (0.11)-0.33 (0.15)-0.97, 0.310.154(6.89 - 12.50)[12.00 - 12.30][12.30 - 12.70][-0.70 - -0.10][5.51, 13.37]Protein32.70 (0.60)31.15 (0.60)1.55 (0.52)-0.70, 3.810.097(29.51 - 40.25)[31.48 - 34.13][30.71 - 31.81][0.55 - 2.32][26.12, 43.51]Total Fat18.70 (0.50)19.88 (0.50)-1.18 (0.40)-2.91, 0.560.099(16.91 - 23.48)[17.41 - 19.50][19.38 - 20.32][-1.98 - -0.73][15.35, 25.95]Vitamin (mg/100g DW)Vitamin E2.20 (0.22)2.91 (0.22)-0.72 (0.31)-2.06, 0.630.148(1.09 - 5.10)[2.07 - 2.42][2.58 - 3.49][-1.40 - -0.16][0, 7.36]AntinutrientLectin (H.U./mg DW)2.28 (0.63)2.46 (0.63)-0.18 (0.90)-4.04, 3.670.855(0.65 - 8.10)[0.82 - 3.38][1.92 - 3.41][-2.59 - 1.31][0, 6.44]Monsanto Company 09-SY-201UPage 290 of 471Table E-10 (continued). Statistical Summaryof Site SFR Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Antinutrient (% DW)Phytic Acid1.90 (0.072)1.88 (0.072)0.023 (0.076)-0.30, 0.350.786(1.42 - 2.27)[1.83 - 2.03][1.72 - 1.97][-0.13 - 0.11][1.35, 2.35]Raffinose0.68 (0.021)0.68 (0.021)-0.0041 (0.029)-0.13, 0.120.901(0.40 - 0.80)[0.62 - 0.71][0.66 - 0.70][-0.064 - 0.051][0.27, 0.87]Stachyose4.06 (0.24)4.10 (0.24)-0.038 (0.35)-1.53, 1.450.922(2.30 - 4.53)[3.88 - 4.41][3.50 - 4.43][-0.56 - 0.91][1.96, 4.41]Trypsin Inhibitor (TIU/mg DW)40.79 (3.57)36.63 (3.57)4.16 (1.82)-3.67, 11.980.149(23.11 - 60.42)[33.98 - 47.66][33.22 - 42.92][0.76 - 6.98][8.75, 63.43]Isoflavone (μg/g DW)Daidzein2481.08 (83.81)2406.95 (83.81)74.13 (58.64)-178.17, 326.420.333(320.54 - 3061.22)[2354.95 - 2565.56][2223.49 - 2565.86][-43.14 - 134.06][0, 3328.03]Genistein1487.13 (52.05)1390.09 (52.05)97.04 (43.02)-88.04, 282.120.152(433.41 - 2301.59)[1433.45 - 1527.94][1254.28 - 1466.21][33.79 - 179.17][0, 2727.33]Monsanto Company 09-SY-201UPage 291 of 471Table E-10 (continued). Statistical Summaryof Site SFR Soybean Seed Amino Acid,Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Glycitein174.86 (13.28)122.44 (13.28)52.42 (18.78)-28.36, 133.210.107(21.67 - 354.30)[152.79 - 196.59][98.05 - 146.12][6.67 - 98.54][0, 376.03]1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standarderror; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 292 of 471Table E-11. Statistical Summary of Combined Site Soybean Forage Fiber and Proximate Content for MON 87705 vs. theConventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Acid Detergent Fiber30.18 (2.20)29.17 (2.23)1.01 (1.64)-2.38, 4.390.543(23.18 - 42.11)[20.04 - 42.14][19.21 - 40.67][-10.04 - 12.39][18.29, 41.02]Ash8.75 (0.22)8.18 (0.22)0.57 (0.23)0.096, 1.050.020(6.76 - 10.40)[7.39 - 10.11][7.21 - 9.32][-0.68 - 1.79][6.78, 9.91]Carbohydrates72.30 (1.20)73.43 (1.21)-1.13 (0.59)-2.36, 0.0970.069(63.74 - 80.60)[68.94 - 78.93][67.88 - 78.99][-4.74 - 5.27][64.45, 80.50]Moisture (% FW)72.93 (1.16)72.22 (1.16)0.70 (0.48)-0.64, 2.040.218(65.80 - 82.00)[70.20 - 81.10][69.40 - 77.50][-1.50 - 7.20][62.26, 83.45]Neutral Detergent Fiber35.79 (2.05)35.85 (2.06)-0.062 (2.14)-6.00, 5.880.978(24.70 - 46.55)[26.12 - 46.30][32.19 - 45.90][-7.01 - 8.75][22.57, 46.52]Protein14.04 (1.13)13.34 (1.13)0.70 (0.55)-0.43, 1.840.213(9.51 - 19.93)[9.25 - 18.10][9.71 - 17.85][-5.20 - 3.75][7.38, 21.27]Monsanto Company 09-SY-201UPage 293 of 471Table E-11 (continued). Statistical Summaryof Combined Site Soybean Forage Fiber and Proximate Content for MON 87705vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber and Proximate (% DW)Total Fat4.91 (0.42)5.14 (0.42)-0.23 (0.38)-1.01, 0.550.549(1.19 - 8.22)[2.29 - 7.01][3.36 - 7.19][-2.23 - 2.80][0, 9.74]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201UPage 294 of 471Table E-12. Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitamin,Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.59 (0.033)1.57 (0.033)0.025 (0.018)-0.026, 0.0750.243(1.34 - 1.78)[1.49 - 1.70][1.43 - 1.66][-0.041 - 0.13][1.25, 1.92]Arginine2.78 (0.11)2.68 (0.11)0.10 (0.036)0.0012, 0.200.048(2.15 - 3.23)[2.43 - 3.16][2.31 - 2.99][-0.062 - 0.39][1.81, 3.62]Aspartic Acid4.08 (0.13)4.00 (0.13)0.075 (0.060)-0.092, 0.240.279(3.37 - 4.76)[3.67 - 4.49][3.51 - 4.43][-0.16 - 0.52][3.02, 5.11]Cystine0.61 (0.0075)0.59 (0.0076)0.022 (0.0075)0.0010, 0.0420.043(0.53 - 0.64)[0.57 - 0.64][0.55 - 0.63][-0.027 - 0.073][0.49, 0.69]Glutamic Acid6.46 (0.24)6.32 (0.24)0.14 (0.12)-0.19, 0.470.300(5.14 - 7.73)[5.72 - 7.19][5.42 - 7.09][-0.32 - 1.02][4.42, 8.48]Glycine1.59 (0.039)1.56 (0.039)0.028 (0.022)-0.033, 0.0890.265(1.30 - 1.79)[1.47 - 1.74][1.41 - 1.67][-0.050 - 0.17][1.19, 1.95]Monsanto Company 09-SY-201UPage 295 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine1.59 (0.033)1.57 (0.033)0.025 (0.018)-0.026, 0.0750.243(1.34 - 1.78)[1.49 - 1.70][1.43 - 1.66][-0.041 - 0.13][1.25, 1.92]Arginine2.78 (0.11)2.68 (0.11)0.10 (0.036)0.0012, 0.200.048(2.15 - 3.23)[2.43 - 3.16][2.31 - 2.99][-0.062 - 0.39][1.81, 3.62]Aspartic Acid4.08 (0.13)4.00 (0.13)0.075 (0.060)-0.092, 0.240.279(3.37 - 4.76)[3.67 - 4.49][3.51 - 4.43][-0.16 - 0.52][3.02, 5.11]Cystine0.61 (0.0075)0.59 (0.0076)0.022 (0.0075)0.0010, 0.0420.043(0.53 - 0.64)[0.57 - 0.64][0.55 - 0.63][-0.027 - 0.073][0.49, 0.69]Glutamic Acid6.46 (0.24)6.32 (0.24)0.14 (0.12)-0.19, 0.470.300(5.14 - 7.73)[5.72 - 7.19][5.42 - 7.09][-0.32 - 1.02][4.42, 8.48]Glycine1.59 (0.039)1.56 (0.039)0.028 (0.022)-0.033, 0.0890.265(1.30 - 1.79)[1.47 - 1.74][1.41 - 1.67][-0.050 - 0.17][1.19, 1.95]Monsanto Company 09-SY-201UPage 296 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Histidine0.96 (0.023)0.94 (0.023)0.027 (0.010)-0.0029, 0.0570.065(0.79 - 1.07)[0.88 - 1.04][0.84 - 1.01][-0.021 - 0.11][0.74, 1.16]Isoleucine1.71 (0.050)1.67 (0.051)0.039 (0.036)-0.064, 0.140.344(1.37 - 2.00)[1.56 - 1.88][1.45 - 1.86][-0.15 - 0.26][1.23, 2.15]Leucine2.73 (0.083)2.69 (0.083)0.044 (0.040)-0.065, 0.150.325(2.26 - 3.14)[2.47 - 3.01][2.37 - 2.94][-0.13 - 0.32][2.06, 3.41]Lysine2.39 (0.053)2.33 (0.053)0.057 (0.025)-0.011, 0.130.080(2.00 - 2.63)[2.19 - 2.55][2.10 - 2.51][-0.051 - 0.25][1.87, 2.81]Methionine0.55 (0.0088)0.53 (0.0089)0.020 (0.011)-0.010, 0.0490.141(0.46 - 0.59)[0.51 - 0.58][0.49 - 0.57][-0.035 - 0.056][0.43, 0.63]Phenylalanine1.82 (0.056)1.80 (0.056)0.019 (0.027)-0.056, 0.0930.523(1.50 - 2.11)[1.64 - 2.00][1.58 - 1.99][-0.16 - 0.22][1.35, 2.31]Monsanto Company 09-SY-201UPage 297 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Proline1.77 (0.057)1.72 (0.057)0.054 (0.030)-0.029, 0.140.145(1.43 - 2.03)[1.59 - 1.95][1.52 - 1.90][-0.046 - 0.27][1.29, 2.21]Serine1.75 (0.059)1.77 (0.059)-0.016 (0.035)-0.087, 0.0560.655(1.55 - 2.05)[1.49 - 1.98][1.45 - 1.94][-0.22 - 0.24][1.44, 2.15]Threonine1.33 (0.030)1.33 (0.030)-0.0031 (0.018)-0.041, 0.0350.867(1.19 - 1.48)[1.20 - 1.45][1.18 - 1.47][-0.13 - 0.11][1.12, 1.53]Tryptophan0.42 (0.011)0.41 (0.012)0.0016 (0.0069)-0.017, 0.0200.831(0.33 - 0.48)[0.37 - 0.46][0.35 - 0.44][-0.028 - 0.046][0.30, 0.50]Tyrosine1.25 (0.029)1.22 (0.029)0.026 (0.014)-0.011, 0.0640.124(1.07 - 1.39)[1.17 - 1.33][1.10 - 1.32][-0.040 - 0.12][0.99, 1.49]Valine1.83 (0.052)1.77 (0.053)0.051 (0.038)-0.059, 0.160.260(1.45 - 2.13)[1.69 - 2.02][1.55 - 1.96][-0.17 - 0.28][1.31, 2.29]Monsanto Company 09-SY-201UPage 298 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)16:0 Palmitic2.36 (0.056)10.83 (0.056)-8.47 (0.055)-8.62, -8.31<0.001(8.78 - 11.51)[2.25 - 2.44][10.51 - 11.08][-8.68 - -8.13][7.62, 12.55]18:0 Stearic3.31 (0.067)4.50 (0.067)-1.19 (0.065)-1.37, -1.01<0.001(3.82 - 7.21)[3.07 - 3.82][4.24 - 4.85][-1.47 - -0.79][2.87, 7.15]18:1 Oleic76.47 (0.59)22.81 (0.59)53.65 (0.22)53.17, 54.13<0.001(20.77 - 27.19)[73.13 - 79.17][21.41 - 25.08][51.71 - 55.05][18.40, 30.22]18:2 Linoleic10.10 (0.39)52.86 (0.39)-42.77 (0.18)-43.17, -42.37<0.001(48.62 - 54.74)[7.85 - 12.42][51.68 - 53.89][-43.74 - -41.38][47.75, 56.46]18:3 Linolenic6.69 (0.28)8.02 (0.28)-1.33 (0.072)-1.53, -1.13<0.001(5.89 - 9.11)[5.55 - 7.81][6.86 - 8.60][-1.80 - -0.59][4.97, 9.93]20:0 Arachidic0.30 (0.0076)0.34 (0.0077)-0.039 (0.0071)-0.059, -0.0190.005(0.28 - 0.54)[0.28 - 0.36][0.31 - 0.36][-0.082 - -0.022][0.22, 0.53]Monsanto Company 09-SY-201UPage 299 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic0.34 (0.013)0.19 (0.013)0.15 (0.0086)0.13, 0.18<0.001(0.15 - 0.22)[0.27 - 0.40][0.15 - 0.21][0.12 - 0.20][0.13, 0.25]22:0 Behenic0.29 (0.0037)0.30 (0.0038)-0.0052 (0.0051)-0.017, 0.00700.346(0.29 - 0.46)[0.28 - 0.33][0.28 - 0.31][-0.029 - 0.020][0.22, 0.47]24:0 Lignoceric0.14 (0.017)0.13 (0.017)0.0046 (0.0046)-0.0084, 0.0180.372(0.056 - 0.21)[0.066 - 0.17][0.067 - 0.16][-0.019 - 0.027][0.030, 0.26]Fiber (% DW)Acid Detergent Fiber17.14 (0.54)16.14 (0.54)1.00 (0.74)-1.06, 3.050.249(12.46 - 21.25)[15.71 - 19.31][13.36 - 18.02][-1.84 - 4.03][12.71, 19.29]Neutral Detergent Fiber18.44 (0.85)17.83 (0.86)0.60 (1.03)-2.25, 3.460.590(12.25 - 20.89)[13.41 - 22.18][14.61 - 21.09][-4.24 - 4.33][12.07, 21.51]Proximate (% DW)Ash6.06 (0.13)6.13 (0.13)-0.072 (0.081)-0.30, 0.150.421(5.64 - 6.82)[5.46 - 6.54][5.48 - 6.55][-0.56 - 0.26][5.26, 7.17]Monsanto Company 09-SY-201UPage 300 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Proximate (% DW)Carbohydrates40.35 (0.86)39.93 (0.86)0.42 (0.37)-0.59, 1.430.317(32.79 - 42.29)[36.69 - 43.52][37.46 - 42.96][-1.08 - 2.00][30.78, 45.86]Moisture (% FW)10.76 (0.37)11.56 (0.37)-0.80 (0.44)-2.02, 0.420.141(6.89 - 12.50)[8.96 - 12.30][10.20 - 12.70][-3.40 - 0.90][5.51, 13.37]Protein35.32 (0.99)34.66 (0.99)0.66 (0.36)-0.33, 1.650.141(29.51 - 40.25)[31.48 - 38.59][30.71 - 37.40][-0.94 - 3.08][26.12, 43.51]Total Fat18.29 (0.39)19.33 (0.39)-1.04 (0.16)-1.39, -0.69<0.001(16.91 - 23.48)[16.55 - 19.50][17.63 - 20.32][-2.16 - -0.20][15.35, 25.95]Vitamin (mg/100g DW)Vitamin E2.83 (0.43)3.27 (0.43)-0.44 (0.17)-0.92, 0.0370.062(1.09 - 5.10)[1.23 - 4.36][1.69 - 4.19][-1.40 - 0.76][0, 7.36]AntinutrientLectin (H.U./mg DW)2.21 (0.40)2.45 (0.41)-0.24 (0.57)-1.57, 1.090.686(0.65 - 8.10)[0.72 - 3.77][0.61 - 5.53][-4.80 - 3.04][0, 6.44]Monsanto Company 09-SY-201UPage 301 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Antinutrient (% DW)Phytic Acid1.82 (0.052)1.85 (0.053)-0.031 (0.041)-0.12, 0.0570.457(1.42 - 2.27)[1.52 - 2.13][1.63 - 2.19][-0.27 - 0.18][1.35, 2.35]Raffinose0.58 (0.029)0.58 (0.029)0.00036 (0.015)-0.042, 0.0430.981(0.40 - 0.80)[0.48 - 0.71][0.50 - 0.70][-0.088 - 0.12][0.27, 0.87]Stachyose3.87 (0.13)3.70 (0.13)0.17 (0.14)-0.22, 0.560.290(2.30 - 4.53)[3.39 - 4.48][3.04 - 4.43][-0.56 - 0.96][1.96, 4.41]Trypsin Inhibitor (TIU/mg DW)38.14 (2.60)37.25 (2.61)0.89 (0.97)-1.74, 3.510.408(23.11 - 60.42)[26.73 - 52.01][27.23 - 49.78][-5.31 - 6.98][8.75, 63.43]Isoflavone (μg/g DW)Daidzein1806.33 (229.35)1794.07 (229.50)12.26 (37.50)-68.88, 93.400.748(320.54 - 3061.22)[1145.72 - 2565.56][1092.43 - 2565.86][-196.53 - 242.28][0, 3328.03]Genistein1160.30 (115.82)1117.27 (115.95)43.04 (24.92)-10.74, 96.810.107(433.41 - 2301.59)[809.79 - 1527.94][751.67 - 1466.21][-104.61 - 208.66][0, 2727.33]Monsanto Company 09-SY-201UPage 302 of 471Table E-12 (continued). Statistical Summary of Combined SiteSoybean Seed Amino Acid, Fatty Acid, Fiber, Proximate,Vitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525)Difference (MON 87705 minus Control)Component (Units)1MON 87705Mean (S.E.)[Range]ControlMean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Glycitein132.85 (12.38)126.86 (12.53)5.98 (12.18)-27.41, 39.380.648(21.67 - 354.30)[49.11 - 196.59][72.10 - 167.04][-61.67 - 98.54][0, 376.03]1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standarderror; CI = Confidence Interval.2With 95% confidence, interval contains 99%of the values expressed inthe population of commercialsoybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201U Page 303 of 471 Table E-13. Literature and ILSI-CCD Ranges for Components in Soybean Forage

and Seed

Forage Tissue/Component 1 Literature Range 2 ILSI-CCD Range 3

Proximate (% dw)

Ash 5.36 – 8.91 6.72 – 10.78 Carbohydrates 62.25 – 72.28 59.8 – 74.7 Moisture (% fw) 68.50 – 78.40 73.5 – 81.6

Protein 16.48 – 24.29 14.38 – 24.71

Total Fat 2.65 – 9.87 1.302 – 5.132

Fiber (% dw) Acid Detergent Fiber (ADF) 23.86 – 50.69 not available Neutral Detergent Fiber (NDF) 19.61 – 43.70 not available

Seed Tissue Component 1 Literature Range 2 ILSI Range 3

Proximates (% dw)

Ash 4.61 – 6.32 3.89 – 6.99 Carbohydrates 32.75 – 40.98 29.6 – 50.2 Moisture (% fw) 6.24 – 11.10 4.7 – 34.4

Protein 34.78 – 43.35 33.19 – 45.48

Total Fat 14.62 – 20.68 8.10 – 23.56

Fiber (% dw) Acid Detergent Fiber (ADF) 9.22 – 26.26 7.81 – 18.61
Neutral Detergent Fiber (NDF) 10.79 – 23.90 8.53 – 21.25

Amino Acids (% dw)

Alanine 1.62 – 1.89 1.51-2.10 Arginine 2.57 – 3.27 2.29-3.40 Aspartic acid 4.16 – 5.02 3.81-5.12 Cystine/Cysteine 0.52 – 0.69 0.37-0.81

Glutamic acid 6.52 – 8.19 5.84-8.20 Glycine 1.59 – 1.90 1.46-2.00

Histidine 0.96 – 1.13 0.88-1.18

Isoleucine 1.59 – 2.00 1.54-2.08

Leucine 2.79 – 3.42 2.59-3.62 Lysine 2.36 – 2.77 2.29-2.84

Methionine 0.45 – 0.63 0.43-0.68 Phenylalanine 1.82 – 2.29 1.63-2.35

Proline 1.83 – 2.23 1.69-2.28

Serine 1.95 – 2.42 1.11-2.48

Threonine 1.44 – 1.71 1.14-1.86 Tryptophan 0.30 – 0.48 0.36-0.50 Tyrosine 1.27 – 1.53 1.02-1.61

Valine 1.68 – 2.09 1.60-2.20Monsanto Company 09-SY-201U Page 304 of 471 Table E-13 (continued). Literature and ILSI-CCD Ranges for Components in Soybean Forage and Seed

1 fw=fresh weight; dw=dry weight; H.U. = hemagglutinating unit; TIU = trypsin inhibitor unit.

2 Lundry et al. (2008). 3 ILSI Crop Composition Database at: http://www.cropcomposition.org

. 4 Padgette et al. (1996).

Conversions: % dw x 10 4 = μ g/g dw; mg/g dw x 10 3 = mg/kg dw; mg/100g dw x 10 = mg/kg dw; g/100g dw x 10 = mg/g dw

Seed Tissue Component 1 Literature Range 2 ILSI-CCD Range 3

Fatty Acids (% dw) (% total) 8:0 Caprylic not available 0.148 – 0.148 10:0 Capric not available not available

12:0 Lauric not available 0.082 – 0.132 14:0 Myristic not available 0.071 – 0.238 14:1 Myristoleic not available 0.121 – 0.125

15:0 Pentadecanoic not available not available

15:1 Pentadecenoic not available not available

16:0 Palmitic 1.44 – 2.35 9.55 – 15.77

16:1 Palmitoleic not available 0.086 – 0.194 17:0 Heptadecanoic not available 0.085 – 0.146 17:1 Heptadecenoic not available 0.073 – 0.087

18:0 Stearic 0.54 – 1.12 2.70 – 5.88

18:1 Oleic 2.87 – 8.82 14.3 – 32.2

18:2 Linoleic 6.48 – 11.6 42.3 – 58.8

18:3 Gamma Linolenic not available not available

18:3 Linolenic 0.72 – 2.16 3.00 – 12.52

20:0 Arachidic 0.04 – 0.7 0.163 – 0.482

20:1 Eicosenoic 0.026 – 0.057 0.140 – 0.350

20:2 Eicosadienoic not available 0.077 – 0.245

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.044 – 0.073 0.277 – 0.595

22:1 Erucic not available not available 24:0 Lignoceric 0.13 – 0.24 4 not available

Vitamins (mg/100g dw)

Vitamin E 1.29 – 4.80 0.19-6.17

Antinutrients Lectin (H.U./mg fw) 0.45 – 9.95 0.09 – 8.46 Trypsin Inhibitor (TIU/mg dw) 20.79 – 59.03 19.59 – 118.68 Phytic Acid (% dw) 0.41 – 1.92 0.63 – 1.96

Isoflavones ( μ g/g dw) (mg/kg dw)

Daidzein 224.03 – 1485.52 60.0 – 2453.5

Genistein 338.24 – 1488.89 144.3 – 2837.2 Glycitein 52.72 – 298.57 15.3 – 310.4

Bio-Actives (% dw)

Raffinose 0.26 – 0.84 0.21 – 0.66 Stachyose 1.53 – 2.98 1.21 – 3.50Monsanto Company 09-SY-201U Page 305 of 471 E.6. Compositional Comparison of Proces sed Fractions from Soybean Seed of

MON 87705 and the Conventional Control

To prepare soybean processed fractions, seed samples were collected from field trials

conducted with MON 87705 and the conventional control at two field sites (Jefferson County, IA and Clinton County, IL) in th e U.S. during the 2007 gr owing season. In addition, 12 commercial conventional soybean re ference varieties were grown separately

at three field sites in the U.S. to determin e a 99% tolerance interval for each component analyzed. The seed samples were processe d into defatted toasted soybean meal (TD soybean meal); refined, bleach ed, and deodorized soybean oil (RBD oil); protein isolate; and crude lecithin fractions. The processe d fractions were analyzed according to the principles outlined in the OECD consensu s document for soybean composition (OECD, 2001). Samples from all three replicates of MON 87705, the control, and the 12 references were analyzed from all plots. The TD soybean meal was analyzed for proximates (moisture, protein, fat, as h and carbohydrates by cal culation), ADF, NDF, amino acids, trypsin inhibitors and phytic acid. The RBD oil was analyzed for fatty acids and vitamin E ( α -tocopherol). The protein isolate fr action was analyzed for amino acids

and moisture. The crude lecithin fr action was analyzed for phosphatides ( α -phosphatidic acid, α phosphatidylcholine, α phosphatidylethanolamine, and α -phosphatidylinositiol).

Compositional analyses were conducted to assess whether the processed fractions prepared from MON 87705 are comparable to those of the conventional control, A3525, which has background genetics similar to MON 87705, but lacks the intentionally modified fatty acid profile and glyphosate to lerance traits. Stat istically significant

differences were determined at the 5% leve l of significance (p<0. 05) using established

statistical methods. In addi tion, 12 commercial conventio nal soybean varieties were grown and analyzed separately to establish a range of natural variability for each analyte, where the range of variability is defined by a 99% tolerance interval for that particular analyte. The methods used for composition analysis of the processed fractions are

summarized in Section E.7. The statis tical analysis comp ared MON 87705 and the conventional control across the two sites (combi ned-site). A summary of the significant differences observed between the processe d fractions prepared from the seed of

MON 87705 and the conventional control is included in Ta ble E-15. A statistical summary of the composition of each processed fraction is included in Tables E-16 to E19. Literature ranges are provided in Ta ble E-20 for soybean meal, Table E-21 for protein isolate, Table E-22 for soybean le cithin, and Table E-23 for soybean oil.

Results show that except for the intende d changes in fatty aci d composition, minor

differences in the levels of less abundant fatty acids in RBD oil and occurrence of low levels of minor fatty acids due to spontan eous isomerisation during the oil refining process, the processed fractions pr oduced from MON 87705 are compositionally equivalent to those of conventional soybean . Further details ar e provided below.

E.6.1. Composition of TD Soybean Meal

Comparison of the composition of TD soybean meal prepared from MON 87705 and the conventional soybean control showed no di fferences (p>0.05) for 21 of the 27 components analyzed. Significant differe nces (p<0.05) were observed for sixMonsanto Company 09-SY-201U Page 306 of 471 components (Table E-15): alanine, glycine, isoleucine, lysine, valine, and NDF. For the statistically significant ami no acid differences, the absolute magnitude of the mean differences from the control were small (<0.1% dw) and the MON 87705 mean values

fell within the 99% tolerance interval for the conventional soybean varieties and also within the range of published values for conventional soyb ean. For the statistically significant difference observed for NDF, the ab solute magnitude of the mean difference from the control was small (1.7% dw) and the MON 87705 mean value was within the

99% tolerance interval for the conventional soybean varieties a nd also within the range of published values for conventional soybean (Tab le E-20). Therefore, these differences were not considered biologica lly relevant from a food and feed safety or nutritional perspective.

As expected, the TD soybean meal from MON 87705 contained a small amount of total fat or oil (0.78% dw), which was also presen t at similar levels (0.86% dw) in the meal from the conventional control (T able E-16). TD soybean meal is expected to contain oil, and accordingly, the National Oil Processors Association has established a minimum oil content (0.5% dw) for defatted soybean meal that meets quality standards and guidelines for soybean meal for domestic and intern ational shipping (NOP A, 2006). Although not analyzed for, the composition of the residual o il present in TD soybean meal is expected be consistent with the intended fatty acid ch anges observed in seed and in RBD oil (see

discussion in Section E.6.2 belo w). Based on these results, apart from the intended fatty acid changes in residual oil, the meal from MON 87705 soybean is considered compositionally equivalent to the meal from conventional soybean.

E.6.2. Composition of RBD Oil

Of the 38 fatty acids analyzed, 21 were excluded from statistical analysis since more than 50% of the observations were below the assay limit of quantitation. Of the 17 fatty acids that could be statistically analyzed, significant differen ces (p<0.05) between MON 87705 and control RBD oil were observed for 13 fatty acids (Table E-15). Four of the 13 differences were expected as they were due to the intended changes in fatty acid levels. Thus, 16:0 palmitic acid levels were signi ficantly lower in MON 87705 (2.49% total FA) than the control (11.59% tota l FA), 18:0 stearic acid levels were significantly lower in MON 87705 (3.22% total FA) than the control (4.47% total FA ); 18:1 oleic acid levels were significantly hi gher in MON 87705 (71.51% total FA ) than the control (23.16% total FA), and 18:2 linoleic acid levels were significan tly lower in MON 87705 (14.41% total FA) than the control (51.08% total FA) (Table E-15).

In addition to the intended changes, six fatty acids were detected in RBD oil that were not detected in seed: 14:0 myristic acid, 16:1 pa lmitoleic acid, 17:0 margaric (heptadecanoic) acid, 17:1 9c heptadecenoic acid, 18:2 other trans isomer fatty acids (excluding 9t,12t linolelaidic), and 18:2 6c,9c, oc tadecadienoic acid. As observe d in seed, levels of several less abundant fatty acids were significantly di fferent (p<0.05) betw een the RBD oil from

MON 87705 and the conventional control. Fo r six of the nine remaining fatty acids where a difference was observed (14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric [heptadecanoic] acid, 20:0 arachidic, 20:1 eicosenoic and 22:0 behenic acids), the absolute magnitude of the differences was small (<0.15% total FA), and the MON 87705Monsanto Company 09-SY-201U Page 307 of 471 mean values fell within the 99% tolerance intervals for the reference soybean varieties and/or within published ranges for conve ntional soybean oil (Codex, 2005).

A significant increase in the level of the mi nor fatty acid 17:1 9c heptadecenoic acid was observed in MON 87705 RBD oil (0.12% total FA ) compared to its level in control RBD oil (0.031% total FA). This is not unexpect ed given the intended shift in fatty acid levels

in MON 87705. The mean level of 17:1 9c heptadecenoic acid in MON 87705 RBD oil was higher than the level in the commercial reference varieties (< 0.06% total FA) and slightly outside reported literature values for soybean oil (ND-0.1% total FA; Codex, 2005). However, 17:1 9c heptad ecenoic acid is known to be present in other vegetables oils such as canola (0.3% tota l FA), corn (0.1% total FA), peanut (0.1% total FA), high oleic safflower (0.1% total FA ), and high oleic sunflower (0 .1% total FA) (Codex, 2005). The presence of 17:1 9c heptadecenoic acid has also been documented in a variety of foods, with levels highest in meats and oils. As shown in the Table E-14, comparable or higher intakes of 17:1 9c heptad ecenoic acid can be achieved in a single serving of tofu, ground beef or soft-spread margarine compar ed to a serving of MON 87705 RBD oil. Therefore, it is concluded that there are no adverse food and feed safety or nutrition

effects associated with the levels of 17:1 9c heptadecenoic acid observed in MON 87705

RBD oil.Monsanto Company 09-SY-201U Page 308 of 471 Table E-14. Estimation of 17:1 9c Heptadecen oic Acid Intake in Selected Foods and

MON 87705

Food Level of 17:1

(g/100 g food) RACC 1

(g) Amount of 17:1 consumed per serving (mg/RACC)

Extra firm nigari tofu (16159) 2 1.085 85 922.0

Ground beef, 80% lean (23572) 2 0.135 85 115.0

Soft-spread margarine, 80% fat, canola based (04684) 2 0.053 14 7.0

MON 87705 0.12 14 17.0

1 Reference Amount Customarily Consumed, which is the U.S. serving size for labeling purposes (21CFR 101.12)

2 Number in parentheses refers to Nutrient Database Number used to identify a particular food within the USDA-ARS food composition database.

The 18:2 other trans fatty acids and 18:2 6c,9c, octa decadienoic acid observed in both

MON 87705 and the conventional control RBD oil were not observed in seed fatty acid analyses and are believed to arise from the spontaneous isomerizati on of unsaturated fatty acids during the oil refining process, pa rticularly during the h eat-requiring deodorization step (Chardigny et al. 1996). The prim ary source of industrially produced trans fatty acids (TFAs) in the human diet is the c onsumption of hydrogenate d vegetable oils in liquid or solid form resulting in food products that may contain in excess of 30% TFA (Chardigny et al. 1996; Ledoux et al

, 2007). Naturally occurring trans fats also arise as a

result of bacterial reduction of unsaturated fatty acids in the gut of ruminant animals, with intake resulting from consumption of meat and dairy products (Chardigny et al. 1996;

Ledoux et al.

, 2007).

The mean level of 18:2 other trans fatty acids in MON 87705 RBD oil was significantly lower than in control RBD oil, and both values were outside the range of the commercial

reference varieties. This is likely due to minor differences in processing methods as the commercial reference soybean varieties were grown and processed separately from MON

87705 and the conventional control. Ho wever, the levels of 18:2 other trans fatty acids in MON 87705 RBD oil were within the rang e of total TFA content in samples of unhydrogenated commercial soybean oil (<3.5%) (Chardigny et al.

, 1996; Ledoux et al.

, 2007; Wolff, 1993). The contribution of MON 87705 RBD oil to overall dietary TFA intake will be minimal relative to commonly e xperienced dietary inta kes, and therefore, are not biologically relevant from a food and feed safety or nutritional perspective.Monsanto Company 09-SY-201U Page 309 of 471 The mean level of 18:2 6c,9c, octad ecadienoic acid in MON 87705 RBD oil was significantly lower than in c onventional control RBD oil, a nd both values were outside the range of the commercial references. This is likely due to minor differences in processing methods as the commercial refe rence soybean varieties were grown and processed separately from MON 87705 and the conventional control. It has been reported that although most fa tty acid double bonds are in the cis configuration, some processes (such as heat treatment) may lead to the migration of double bonds from their naturally occurring positions in the carbon chain, leading to an increase in the levels of other cis isomers (Ledoux et al., 2007).

The RBD oil was also analyzed for vitamin E levels which were not significantly different (p>0.05) between MON 87705 and conventional control RBD oil.

In summary, except for intended changes in fa tty acid composition, minor differences in the levels of less abundant fatty acids and occurrence of low levels of minor fatty acids due to spontaneous isomerisation during the oil refining process, the RBD oil from MON 87705 is considered compositionally equivale nt to oil from conventional soybean.

E.6.3. Composition of Soybean Protein Isolate

There were no statistically significant differences (p<0 .05) between MON 87705 and the conventional control for components measured in the protein isolate fraction. Statistical summary data on the composition of the soybean protein isolate frac tion is included in

Table E-18. Based on these results, the protein isolate prepared from MON 87705 is considered compositionally equivalent to pr otein isolate from c onventional soybean.

E.6.4. Composition of Crude Lecithin

There were no statistically significant differences (p<0 .05) between MON 87705 and the conventional control for phosphatides of crude l ecithin. Statistical summary data on the composition of the crude lecithin fraction is included in Table E-19. Based on these results, the MON 87705 soybean lecithin is considered compositionally equivalent to conventional soybean lecithin.

E.6.5. Compositional Equivalence of MON 87705 and Conventional Soybean

Processed Fractions

The processed fractions, TD s oybean meal, RBD oil, protein isolate and crude lecithin, were analyzed according to the principles outlined in the OECD consensus document for soybean composition (OECD, 2001) . There were no statistical ly significant differences (p<0.05) observed between MON 87705 and the conventional control (A3525) for the components measured in protein isolates or crude lecithin. Si gnificant differences (p<0.05) were observed for six (alanine, glycine, isoleucine , lysine, valine, and NDF) of 27 components measured in TD soybean meal; however, the magnitude of the differences

from the conventional control was small a nd the MON 87705 mean values were within

the 99% tolerance interval for the conventiona l reference soybean vari eties and/or within the range of published values for conventiona l soybean meal, indicat ing that they were not biologically meaningful. The low levels of oil (0.78% dw, as total fat) present in TD soybean meal are expected to reflect the intended fatty acid changes observed in seed.Monsanto Company 09-SY-201U Page 310 of 471 As expected, and consistent with results obtained for seed fatty acid levels, the intended fatty acid changes were observed in the RBD oil fraction from MON 87705. As observed in seed, levels of several less abundant fatty acids were significantl y different (p<0.05)

between the RBD oil from MON 87705 and the conventional control. Of the 17 fatty

acids that could be statistical ly analyzed in RBD oil, significant differences (p<0.05)

between MON 87705 and the conventional contro l 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 (16:0 palmitic, 18:0 palmitic, 18: 1 oleic and 18:2 linoleic). For six of the nine remaining differences ( 14:0 myristic acid, 16:1 palm itoleic acid, 17:0 margaric [heptadecanoic] acid, 20:0 arachidic, 20:1 eicosenoic and 22:0 behenic acids), the magnitude of the differences was small (<0.15% total FA), and the MON 87705 mean

values fell within the 99% tolerance interval s for the reference varieties and/or within published ranges for conventional RBD s oybean oil (Codex, 2005, Table E-23).

The remaining three differences were for 17:1 9c heptadecenoic acid, 18:2 trans isomer fatty acids (excluding linolelai dic), and 18:2 6c,9c, octadeca dienoic acid. A significant increase in the level of the minor fatty aci d 17:1 9c heptadecenoic acid was observed in MON 87705 compared to conventio nal control RBD oil. This is not unexpected given the intended shift in fatty acid levels in MON 87705. The mean level of 17:1 9c heptadecenoic acid in MON 87705 was outside the range of values obtained for the RBD oil from commercial reference soybean varie ties. However, 17:1 9c heptadecenoic acid is present at similar or higher levels in a vari ety of oils (canola, co rn, peanut, high oleic safflower, and high oleic sunflower) and f oods (tofu, ground beef, and soft-spread margarine). Therefore, it is concluded that there are no a dverse food and feed safety or

nutrition effects associated with the levels of 17:1 9c heptadecenoic acid observed in MON 87705 soybean oil. The minor fatty acids, 18:2 other trans (excluding linolelaidic) and 18:2 6c,9c, octadecadienoic acid, are believed to arise from the spontaneous isomerization of unsaturated fatty ac ids during the oil refining process.

Therefore, this compositional assessment supports the conclusion that, except for the intended changes in fatty acid composition, minor differences in the levels of less abundant fatty acids and occurrence of low leve ls of minor fatty acids due to spontaneous isomerisation during the oil refining proces s, the processed fractions produced from MON 87705 are compositionally equivalent to those of conventional soybean.Monsanto Company 09-SY-201UPage 311 of 471Table E-15. Summary of Differences (p<0.05)for the Comparison of Soybean Processed Fraction Component Levels forMON 87705 vs. the Conventional Control (A3525) and Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMean Difference(% of A3525)Signif.(p-Value)TestRangeCommercial Range2(99% Tolerance Interval)2,3Meal Amino Acid (% DW)Alanine 2.26 2.33 -3.06 0.019 2.22 - 2.29 2.18 – 2.49(1.87, 2.74)Glycine 2.28 2.34 -2.44 0.023 2.26 - 2.33 2.19 – 2.46(1.91, 2.68)Isoleucine 2.45 2.53 -3.170.006 2.40 - 2.51 2.36 – 2.71(2.03, 3.06)Lysine 3.25 3.34 -2.49 0.030 3.22 - 3.29 3.07 – 3.48(2.65, 3.85)Valine 2.55 2.64 -3.47 0.003 2.51 - 2.60 2.48 – 2.91(2.07, 3.26)Meal Fiber (% DW)Neutral DetergentFiber 8.55 6.81 25.47 0.0168.05 - 8.96 6.20 – 10.58(2.19, 13.59)RBD Oil Fatty Acid (% Total FA)14:0 Myristic 0.031 0.090 -65.15<0.001 0.031 - 0.032 0.066 – 0.11(0.024, 0.14)16:0 Palmitic 2.49 11.59 -78.50<0.001 2.36 - 2.69 9.22 – 11.96(7.75, 13.82)16:1 Palmitoleic 0.13 0.11 22.100.012 0.12 - 0.14 0.072 – 0.11(0.044, 0.14)17:0 Margaric [Heptadecanoic] 0.036 0.10-65.27 0.002 0.031 - 0.048 0.047 – 0.10(0.0082, 0.16)17:1 9c Heptadecenoic 0.12 0.031279.62 0.006 0.092 - 0.14 0 < 0.02not calculated18:0 Stearic 3.22 4.47 -28.05<0.001 3.00 - 3.40 3.58 – 5.00(1.83, 6.48)Monsanto Company 09-SY-201UPage 312 of 471Table E-15 (continued). Summary of Differences (p<0.05) for the Comparison of SoybeanProcessed Fraction ComponentLevels for MON 87705 vs. the ConventionalControl (A3525) and Reference VarietiesMean Difference(MON 87705 minus Control)Component (Units)1MON 87705MeanControlMeanMean Difference(% of A3525)Signif.(p-Value)TestRangeCommercial Range2(99% Tolerance Interval)2RBD Oil Fatty Acid (% Total FA)18:1 Oleic 71.51 23.16 208.71 <0.001 69.30 - 73.01 21.10 – 31.19(11.72, 37.78)18:2 6c,9c Octadecadienoic 0.20 0.65 -69.86 <0.001 0.16 - 0.24 0.031 – 0.074(0, 0.13)18:2 Linoleic 14.41 51.08 -71.78<0.001 12.25 - 17.39 47.74 – 53.88(42.34, 61.19)18:2 Other Trans 0.18 0.63 -70.92 <0.001 0.14 - 0.23 0 < 0.02Not calculated20:0 Arachidic 0.29 0.36 -19.970.001 0.27 - 0.31 0.28 – 0.43(0.13, 0.59)20:1 11c Eicosenoic 0.33 0.19 73.17<0.001 0.29 - 0.37 0.18 – 0.27(0.066, 0.37)22:0 Behenic 0.31 0.35 -9.740.001 0.30 - 0.35 0.30 – 0.50(0.11, 0.70)1 DW = dry weight; FA = fatty acid.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negativelimits were set to zero.Monsanto Company 09-SY-201U Page 313 of 471 E.7. Methods and Materials for Composit ional Analysis of Processed Fractions

MON 87705 and a conventional soybean cont rol (A3525) were grown at two U.S. locations in 2007. MON 87705 was grown from seed lots GLP-0704-18516-S and GLP0705-18715-S, and the conventional soybean control was grown from seed lots GLP0704-18512-S and GLP-0705-18716-S. The c onventional soybean control has background genetics representative of MON 87705 but does not contain cp4 epsps or the

FATB1-A and FAD2-1A gene segments. In additi on, 12 commercial conventional soybean varieties produced at three locations in separate U.S. field trials in 2007 were included for the generation of a 99% toleran ce interval. The conve ntional varieties, locations, and seed lot numbers are listed below:

Material Name Starting Seed Lot No. Field Site

Anand GLP-0705-18678-S AR

UA4805 GLP-0705-18679-S AR

Ozark GLP-0705-18680-S AR

Delta & Pine 5989 GLP-0705-18681-S AR

H437 GLP-0705-18686-S IL

NK S38-T8 GLP-0705-18687-S IL

LG C3540 GLP-0705-18688-S IL

HS38C60 GLP-0705-18689-S IL

NuPride 2954 GLP-0705-18682-S NE

NC+ 2A86 GLP-0705-18683-S NE

Pioneer 92B72 GLP-0705-18684-S NE

NK 25-J5 GLP-0705-18685-S NE

E.7.1. Characterization of the Materials

The identities of MON 87705, the conventio nal soybean control, and conventional reference soybean varieties were verified pr ior to use by confirming the chain-of-custody documentation of the samples from the field cooperators. The seed samples from MON 87705 and the conventional soybean cont rol were further characterized by an event-specific PCR analysis of the DNA extrac ted from the seed to confirm the presence

or absence of MON 87705.Monsanto Company 09-SY-201U Page 314 of 471 E.7.2. Field Production of the Samples

Seed of the MON 87705 and the conventional soybean control were collected from two plots at each of two sites across the U.S. during the 2007 growing season. The field sites were in Clinton County, IL and Jefferson Co unty, IA. The reference varieties were produced separately from one pl ot grown at one of three site s in the U.S. in 2007. The reference soybean varieties were planted at field sites in Jacks on County, AR, Clinton County, IL, and York County, NE. All the reference varieties were grown under normal agronomic field conditions for their respectiv e geographic regions. Seed was shipped at ambient temperature from all production locati ons to Monsanto Company, St. Louis, MO, USA. A subsample for compositional analyses was obtained from each seed sample. The seed subsamples were ground and stored in a fr eezer set to maintain -20° C at Monsanto Company (St. Louis, MO) prior to transfer to Covance Laboratories, Inc. (Madison, WI). The labels on the samples shipped to Covance laboratories Inc. list ed the Monsanto study number, crop and sample type, production site and plot number, storage conditions, unique sample ID, lot or source number, material name, container type, and contact name.

Additionally, a subsample of each MON 87705, conventional control and reference seed sample was shipped from Monsanto Compan y (St. Louis, MO) to GLP-Technologies (GLP-T) in Navasota, Texas, for processing into TD soybean meal, refined, RBD oil, protein isolate and crude lecithin. A subs ample for use in compositional analysis was obtained from each processed sample generate d at GLP-T, and was shipped on dry ice to Monsanto Company (St. Louis, MO). The samp le containers were relabeled at Monsanto with the following informati on: Monsanto study number, processing site, sample identifier, lot number, crop and sample type, material name, container type, contact name, and storage conditions, and then shipped ove rnight on dry ice to Covance Laboratories Inc. (Madison, WI) for analyses.

E.7.3. Compositional Analysis Me thods for Processed Fractions

The compositional analysis methods for ADF, amino acids, ash, carbohydrates, fat, isoflavones, lectin, moisture, NDF, phytic acid, protein, and vitamin E were the same as

described in Section E.6. Methods for anal ytes that had differe nt standards and/or

detection limits and the method for phosphatides, are described below.

E.7.3.1. Fatty Acid Profile with Trans Fat by GC

The lipid was extracted, saponified with 0.5N methanolic sodium hydroxide, and methylated with 14% BF 3 -methanol. The resulting methyl es ters of the fatty acids were extracted with heptane. An internal standard was added prior to the lipid extraction. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation. Th e limit of quantitation was 0.0200% – 0.0600% depending

on the matrix.

Reference StandardsMonsanto Company 09-SY-201U Page 315 of 471 • Nu Chek Prep GLC Reference Standard Hazelton No. 1, Lot Number JY19-R,

100%*

• Nu Chek Prep GLC Reference Standard Hazelton No. 2, Lot Number M13-O,

100%*

• Nu Chek Prep GLC Reference Standard Hazelton No. 3, Lot Number MA18-S,

100%*

• Nu Chek Prep GLC Reference Standard Hazelton No. 4, Lot Number JY19-R,

100%*

• Nu Chek Prep Methyl Gamma Linolenat e, used as 100% , Lot Number U-63MJY12-R

• Nu Chek Prep Methyl Tridecanoate, used as 100%, Lot Number N-13M-F5-S

• Nu Chek Prep Methyl Butyrate, used as 100%, Lot Number N-4M-J20-R

• Nu Chek Prep Methyl Hexanoate, us ed as 100%, Lot Number N-6M-A25-R

• Nu Chek Prep Methyl Erucate, us ed as 100%, Lot Number U-79M-AU3-Q

• Nu Chek Prep Methyl Lignocerate, us ed as 100%, Lot Number N-24M-F5-S

• Nu Chek Prep Methyl Docosapentae noate, used as 100%, Lot Number U101M-F18-S

• Nu Chek Prep Methyl Docosahexaenoa te, used as 100%, Lot Number U-84MD24-R

• Nu Chek Prep Methyl Eicosapentaenoa te, used as 100%, Lot Number U-99MD14-R

• Cayman Chemicals Steariodonic Acid Me thyl Ester, used as 100%, Lot

Number 186208-192001 and 186208-192002

• Nu Chek Prep Methyl Elaidate, us ed as 100%, Lot Number U-47M-JA18-R

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-F27-R

• Nu Chek Prep Methyl Nervonate, us ed as 100%, Lot Number U-88M-O19-R

• Nu Chek Prep Methyl Palmitelaidate, used as as 100%, Lot Number U-41MO26-R

• Monsanto Mono Trans SDA, 99%, Lot Number GLP-0804-19309-A

• Monsanto Alpha Linolenic Acid, us ed as 100%, Lot Number GLP-0804-19308-

A

• Monsanto 9c, 15c Octadecadienoate (Omnisoy), used as 100%, Lot Number

GLP-0802-19168-A

• Larodan Methyl 6(z), 9(z )-Octadecadienoate, used as 99.6%, Lot Number LS113

• Monsanto Omnisoy C17:1 Methyl 9-ci s-Heptadecenoate, used as 99%, Lot

Number GLP-0806-19436-A

* Overall purity of the sum of the mixture components

Literature ReferenceMonsanto Company 09-SY-201U Page 316 of 471 AOCS. 1997. Method Ce 1-62 in Official Methods and Recommended Practices of the

AOCS, 5th ed. American Oil Chem ists' Society, Champaign, IL.

E.7.3.2. Isoflavones Analysis

The sample was extracted using a solution of hydrochloric acid and reagent alcohol heated on steam baths or hot plates. The ex tract was brought to volume, diluted, and centrifuged. An aliquot of the supernatan t was placed onto a C18 solid-phase extraction column. Unwanted components of the matrix were rinsed off with 20% methanol and

then the isoflavones were eluted with 80% methanol. The sample was analyzed on a HPLC system with ultraviolet detection and was compared to an external standard curve of known standards for quantitation. The limit of quantitation for each component was

10.0 μ g/g.

Reference Standards

• Indofine, Daidzein, 99%, Lot Number 020508146

Indofine, Genistein, > 99%, (used as 100%), Lot Number 0309074

Indofine, Glycitein, 99%, Lot Number 0704034

Literature References

Seo, A. and C.V. Morr. 1984. Improved high-performance liquid chromatographic analysis of phenolic acids a nd isoflavonoids from soybean protein products. Journal of Agricultural and Food Ch emistry, 32(3):530-533.

Pettersson, H., and K.H. Kie ssling. 1984. Liquid Chromatogr aphic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official Analytical Chemists Journal, 67(3):503-506.

E.7.3.3. Phosphatides

The sample was extracted with a 98% CHCl 3 2% MeOH solvent. The extract was then analyzed on an HPLC system equipped with an evaporative light-scattering detector (ELSD). A calibration curve was used for qua ntification. The LOQs for these assays were as follows: L-alpha-Phosphatidic Ac id 0.70%, L-alpha-Phosphatidylcholine 1.30%, L-alpha-Phosphatidylethanolamine 1.30%, a nd L-alpha-Phosphatidy linositol 0.70%.

Reference Standards

• (PA) – Avanti Polar Lipids, L-alpha-Phos phatidic Acid (sodium salt), 100%,

Lot Numbers SPA-19 and SPA-20.

• (PC) – Avanti Polar Lipids, L-al pha-Phosphatidylcholine, 100%,

Lot Numbers PPC-116f and PPC-117Monsanto Company 09-SY-201U Page 317 of 471 • (PE) – Avanti Polar Lipids, L-alpha -Phosphatidylethanolamine, 100%,

Lot Numbers PPE-133 and PPE-133c.

• (PI) – Avanti Polar Lipids, L-alpha-Phos phatidylinositol (Sodium salt), 100%,

Lot Numbers PPI-151 and PPI-154.

Literature Reference

AOCS Official Met hod Ja 7b-91. 1997. Determination of Lecithin Phospholipids by

HPLC.

E.7.3.4. Raffinose and Stachyose

The sample was extracted with deionized water and the extract treated with a hydroxylamine hydrochloride solution in pyridine, containing phenyl- β -D-glucoside as an internal standard. The resulting oxim es were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization detector. The limit of quantitation was

0.0500%.

Reference standards

• Sigma, Raffinose Pentahydr ate, 99% / 84.0% after correction for degree of hydration, Lot Number 035K1371

• Sigma, Stachyose, 98% / 96.4% after co rrection for degree of hydration, Lot

Number 065K3775

Literature References

Brobst, K. M. 1972. Gas-Liquid Chromatogr aphy of Trimethylsilyl Derivatives in Methods in Carbohydrate Chemistry, Vo l. 6. Academic Press, New York.

Mason, B.S., and H.T. Slover. 1971. A Gas Chromatographic Method for the Determination of Sugars in Foods. Journa l of Agricultural and Food Chemistry, 19(3):551-554.Monsanto Company 09-SY-201U Page 318 of 471 E.7.3.5. Trypsin Inhibitor

The sample was ground and defatted with petr oleum ether. A sample of matrix was

extracted for three hours with 0.01 N sodium hydroxide. Vary ing aliquots of the sample suspension were exposed to a know n amount of trypsin and benzoy1-DLarginine~p~nitroanilide hydrochl oride. The sample was allowed to react for 10 minutes at 37°C. After 10 minutes, the reaction wa s halted by the addition of acetic acid. The solution was centrifuged, then the absorban ce was determined at 410 nm. Trypsin inhibitor activity was determined by photometri cally measuring the inhibition of trypsin’s reaction with benzoyl-DL-arginine~p~nitr oanilide hydrochloride. The limit of quantitation was 1.00 Trypsin Inhibitor Units (TIU)/mg.

Literature Reference

AOCS. 1997. Method Ba 12-75 in Official Methods and Reco mmended Practices of the American Oil Chemists’ Society. AOCS Press, Champaign, IL.

E.8 Data Processing

After compositional analyses were performed at Covance Laboratories Inc., data spreadsheets were forwarded to Monsanto Comp any. The data were reviewed, formatted, and sent to Certus International, Inc. for st atistical analysis. A statistical sub-report was generated by Certus Internati onal, Inc. and sent to Mons anto Company. In all, 27 components were analyzed in m eal, 39 in RBD oil, 19 in pr otein isolates, and four in lecithin. The following RBD oil components we re excluded from statistical analysis: 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:1 myristoleic acid , 15:0 pentadecanoic acid, 15:1 10c pentadecenoi c acid, 16:1 palmitelaidic, 18: 1 elaidic acid, 18:2 9c,15c octadecadienoic acid, 18:2 linolelaidic acid, 18:3 ga mma linolenic acid, 18:4 6c,9c,12c,15t trans-SDA, 18: 4 stearidonic acid, 20:2 11c, 14 c eicosadienoic acid, 20:3 11c,14,17c eicosatrienoic acid, 20:4 ar achidonic acid, 20:5 5c,8c,11c,14c,17c eicosapentaenoic acid, 22:1 erucic acid, 22: 5 7c,10c,13c,16c,19c docosapentaenoic acid, 22:6 4c,7c,10c,13c,16c,19c docosehexaenoic ac id, and 24:1 nervonic acid. The LOQ for the oil fatty acid method was 0.0600% fresh weight.

The following observations were below the LOQ: five observations for trypsin inhibitor in meal; eight observations for 14:0 myri stic acid, seven observations for 17:0 heptadecenoic acid, and eight observations for 17:1 9c he ptadecenoic acid in RBD oil; four observations for L-alpha-phosphatidic acid and two observations for L-alphaphosphatidylethanolamine in lecithin. These obs ervations were assigned a value equal to half the LOQ for the respective assay.

The data were assessed for potential outli ers using a studentiz ed PRESS residuals

calculation. No values in the da ta set were removed as outliers.Monsanto Company 09-SY-201U Page 319 of 471 E.9. Statistical Methodology

The compositional components for MON 87705 and the conventional soybean control were statistically analyzed acr oss all sites using a mixed-mode l analysis of variance. The combined-site analyses used the model: (1) Y ij = U + T i + B j + e ij,

where Y ij = unique individua l observation, U = overall mean, T i = substance effect, B j = random replication block effect, and e ij = residual error. For the processed fractions compositional components, MON 87705 was co mpared to the conventional soybean

control.

A range of observed values from the referen ce soybean varieties was determined for each analytical component. Additionally, the reference soybean varieties’ data were used to develop population tolerance intervals. A tole rance interval is an interval that one can claim, with a specified degree of confidence, co ntains at least a speci fied proportion, p, of an entire sampled population for the parameter measured. For each compositional component, 99% tolerance interv als were calculated that are expected to contain, with 95% confidence, 99% of the quantities expresse d in the population of conventional reference soybean varieties (Ridley et al ., 2002; George et al., 2004). Each tolerance interval estimate was based upon one observation per unique reference variety. A single replicate from each unique reference variety was analyzed for inclusion in tolerance interval calculations. Because negative quant ities are not possible, calculated negative

lower tolerance bounds were set to zero. SA S programming was used to generate all summary statistics and perform all analyses (SAS Institute 2002-2003). Report tables present p-values from SAS as either <0. 001 or the actual value truncated to three

decimals.Monsanto Company 09-SY-201UPage 320 of 471Table E-16. Comparison of Amino Acid, Fiber, Proximate, and AntinutrientContent from MON 87705 andConventional Control (A3525) Soybean Meal Processed from a 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercialRange[99% Tolerance Int.2]Amino Acid (% DW)Alanine 2.26 (0.021) 2.33 (0.021) -0.071 (0.016) -0.12, -0.022 0.019 2.18 - 2.49[2.22 - 2.29] [2.26 - 2.38] [1.87, 2.74]Arginine 4.22 (0.074) 4.31 (0.074) -0.091 (0.075) -0.33, 0.15 0.312 3.83 - 4.84[4.01 - 4.37] [4.17 - 4.49] [2.79, 5.46]Aspartic Acid 5.95 (0.068) 6.16 (0.068) -0.21 (0.066) -0.42, 0.0058 0.053 5.69 - 6.63[5.83 - 6.14] [5.99 - 6.32] [4.80, 7.35]Cystine 0.82 (0.028) 0.81 (0.028) 0.0016 (0.019) -0.059, 0.062 0.938 0.69 - 0.82[0.75 - 0.90] [0.78 - 0.87] [0.56, 0.94]Glutamic Acid 9.43 (0.11) 9.78 (0.11) -0.35 (0.14) -0.79, 0.091 0.086 9.05 - 10.63[9.17 - 9.70] [9.48 - 9.98] [7.62, 11.74]Glycine 2.28 (0.018) 2.34 (0.018) -0.057 (0.013) -0.099, -0.015 0.023 2.19 - 2.46[2.26 - 2.33] [2.29 - 2.38] [1.91, 2.68]Histidine 1.40 (0.019) 1.44 (0.019) -0.041 (0.016) -0.093, 0.012 0.089 1.29 - 1.62[1.38 - 1.43] [1.39 - 1.50] [0.99, 1.88]Isoleucine 2.45 (0.021) 2.53 (0.021) -0.080 (0.012) -0.12, -0.043 0.006 2.36 - 2.71[2.40 - 2.51] [2.50 - 2.58] [2.03, 3.06]Monsanto Company 09-SY-201UPage 321 of 471Table E-16 (continued). Comparisonof Amino Acid, Fiber, Proximate, and Antinutrient Content from MON 87705and Conventional Control (A3525) Soybean MealProcessed from a 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Amino Acid (% DW)Leucine 4.02 (0.038) 4.14 (0.038) -0.11 (0.038) -0.23, 0.0060 0.056 3.92 - 4.39[3.94 - 4.13] [4.04 - 4.22] [3.41, 4.84]Lysine 3.25 (0.019) 3.34 (0.019) -0.083 (0.022) -0.15, -0.015 0.030 3.07 - 3.48[3.22 - 3.29] [3.28 - 3.39] [2.65, 3.85]Methionine 0.82 (0.024) 0.84 (0.024) -0.021 (0.025) -0.10, 0.057 0.452 0.71 - 0.80[0.76 - 0.89] [0.79 - 0.89] [0.65, 0.86]Phenylalanine 2.70 (0.030) 2.79 (0.030) -0.093 (0.032) -0.19, 0.0074 0.060 2.68 - 3.02[2.65 - 2.78] [2.71 - 2.86] [2.28, 3.29]Proline 2.65 (0.040) 2.76 (0.040) -0.11 (0.037) -0.23, 0.0099 0.061 2.48 - 2.87[2.60 - 2.76] [2.65 - 2.85] [2.15, 3.08]Serine 2.64 (0.037) 2.73 (0.037) -0.093 (0.050) -0.25, 0.065 0.157 2.50 - 2.84[2.58 - 2.69] [2.61 - 2.81] [2.25, 3.02]Threonine 2.06 (0.027) 2.13 (0.027) -0.070 (0.031) -0.17, 0.028 0.106 1.89 - 2.09[1.99 - 2.09] [2.05 - 2.18] [1.73, 2.24]Tryptophan 0.60 (0.013) 0.60 (0.013) -0.0030 (0.013) -0.045, 0.039 0.835 0.55 - 0.64[0.57 - 0.62] [0.57 - 0.64] [0.47, 0.72]Monsanto Company 09-SY-201UPage 322 of 471Table E-16 (continued). Comparisonof Amino Acid, Fiber, Proximate, and Antinutrient Content from MON 87705and Conventional Control (A3525) Soybean MealProcessed from a 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercialRange[99% Tolerance Int.2]Amino Acid (% DW)Tyrosine 1.83 (0.023) 1.85 (0.023) -0.027 (0.021) -0.093, 0.039 0.279 1.55 - 1.97[1.78 - 1.85] [1.80 - 1.91] [1.29, 2.26]Valine 2.55 (0.020) 2.64 (0.020) -0.092 (0.010) -0.13, -0.058 0.003 2.48 - 2.91[2.51 - 2.60] [2.61 - 2.69] [2.07, 3.26]Fiber and Proximate (% DW)Acid Detergent Fiber 6.54 (0.26) 5.94 (0.26) 0.59 (0.37) -0.30, 1.49 0.156 4.34 - 8.07[5.96 - 6.89] [5.34 - 6.70] [1.27, 10.32]Neutral Detergent Fiber 8.55 (0.26) 6.81 (0.26) 1.74 (0.36) 0.59, 2.88 0.016 6.20 - 10.58[8.05 - 8.96] [6.26 - 7.50] [2.19, 13.59]Ash 6.99 (0.17) 7.29 (0.17) -0.29 (0.097) -0.60, 0.014 0.056 6.36 - 7.45[6.74 - 7.30] [6.88 - 7.76] [5.71, 8.17]Carbohydrates 39.31 (0.71) 37.69 (0.71) 1.62 (0.59) -0.25, 3.49 0.070 32.34 - 41.36[37.73 - 40.04] [35.68 - 39.61] [27.18, 48.54]Moisture (% FW) 3.33 (1.41) 6.86 (1.41) -3.53 (2.00) -8.41, 1.35 0.127 3.74 - 12.65[2.72 - 3.91] [3.94 - 12.50] [0, 21.82]Monsanto Company 09-SY-201UPage 323 of 4711DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval; ND = not detected; NC = not calculated.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial varieties. Negative limits were set to zero.Table E-16 (continued). Comparisonof Amino Acid, Fiber, Proximate, and Antinutrient Content from MON 87705and Conventional Control (A3525) Soybean MealProcessed from a 2007 U.S. Field ProductionDifference (MON 87705 minusControl)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Protein 52.91 (0.59) 54.16 (0.59) -1.24 (0.63) -3.24, 0.76 0.142 51.40 - 59.42[52.01 - 54.17] [52.52 - 55.85] [44.28, 64.08]Total Fat 0.78 (0.084) 0.86 (0.084) -0.079 (0.10) -0.41, 0.25 0.495 0.65 - 1.72[0.61 - 1.04] [0.69 - 1.00] [0, 2.12]Antinutrient (% DW)Phytic Acid 1.37 (0.033) 1.47 (0.033) -0.10 (0.033) -0.21, 0.0047 0.055 1.06 - 1.53[1.27 - 1.43] [1.44 - 1.54] [0.67, 1.90]Trypsin Inhibitor (TIU/mg DW) 2.15 (0.59) 1.41 (0.59) 0.74 (0.83) -1.29, 2.77 0.405 ND[0.52 - 4.09] [0.54 - 2.07] NCMonsanto Company 09-SY-201UPage 324 of 471Table E-17. Comparison of Fatty Acids and Vitamin E from MON 87705 and Conventional Control (A3525)Soybean RBD Oil Processed from the 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercialRange[99% Tolerance Int.2]RBD Oil Fatty Acid (% Total FA)14:0 Myristic 0.031 (0.0017) 0.090(0.0017) -0.059 (0.0025) -0.065,-0.053 <0.001 0.066 - 0.11[0.031 - 0.032] [0.086 - 0.097] [0.024, 0.14]16:0 Palmitic 2.49 (0.087) 11.59(0.087) -9.10 (0.087) -9.37, -8.82 <0.001 9.22 - 11.96[2.36 - 2.69] [11.36 - 11.83] [7.75, 13.82]16:1 Palmitoleic 0.13 (0.0044) 0.11 (0.0044) 0.023 (0.0044) 0.0094, 0.037 0.012 0.072 - 0.11[0.12 - 0.14] [0.096 - 0.11] [0.044, 0.14]17:0 Margaric 0.036 (0.0070) 0.10 (0.0070) -0.067(0.0073) -0.091, -0.044 0.002 0.047 - 0.10[0.031 - 0.048] [0.085 - 0.12] [0.0082, 0.16]17:1 9c Heptadecenoic 0.12 (0.0090) 0.031 (0.0090) 0.088 (0.013) 0.047, 0.13 0.006 ND[0.092 - 0.14] [0.031 - 0.031] NC18:0 Stearic3.22 (0.072) 4.47 (0.072) -1.25 (0.060) -1.44, -1.06 <0.0013.58 - 5.00[3.00 - 3.40] [4.33 - 4.57][1.83, 6.48]18:1 Oleic71.51 (0.85) 23.16 (0.85) 48.35 (1.21) 45.39, 51.30 <0.00121.10 - 31.19[69.30 - 73.01] [21.44 - 25.54][11.72, 37.78]18:2 6c,9c Octadecadienoic0.20 (0.017) 0.65 (0.017) -0.46 (0.024) -0.52, -0.40 <0.0010.031 - 0.074[0.16 - 0.24] [0.63 - 0.69][0, 0.13]Monsanto Company 09-SY-201UPage 325 of 471Table E-17 (continued). Comparison of Fatty Acidsand Vitamin E from MON 87705 and Conventional Control(A3525) Soybean RBD Oil Processed from the 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper) p-ValueCommercial(Range)[99% Tolerance Int.2]RBD Oil Fatty Acid (% TotalFA)18:2 Linoleic 14.41 (0.92) 51.08 (0.92) -36.67 (1.18) -40.44, -32.90 <0.001 47.74 - 53.88[12.25 - 17.39] [50.02 - 52.06] [42.34, 61.19]18:2 Other Trans 0.18 (0.017) 0.63 (0.017) -0.44 (0.024) -0.50, -0.39 <0.001 ND[0.14 - 0.23] [0.60 - 0.66] NC18:3 9c,12c,15tOctadecatrienoic0.92 (0.073) 0.98 (0.073) -0.059 (0.045) -0.20, 0.085 0.284 0.031 - 0.13[0.81 - 1.01] [0.80 - 1.19] [0, 0.22]18:3 Linolenic 4.76 (0.38) 5.13 (0.38) -0.37 (0.15) -0.87, 0.12 0.094 5.07 - 9.34[4.08 - 5.46] [4.21 - 5.96] [1.19, 12.72]18:3 Other 18:3 Trans 0.92 (0.067) 0.93 (0.067) -0.011 (0.045) -0.15, 0.13 0.815 0.031 - 0.092[0.81 - 1.01] [0.78 - 1.16] [0, 0.17]20:0 Arachidic 0.29 (0.0088) 0.36 (0.0088) -0.072 (0.0060) -0.091, -0.053 0.001 0.28 - 0.43[0.27 - 0.31] [0.34 - 0.38] [0.13, 0.59]20:1 Eicosenoic 0.33 (0.016) 0.19 (0.016) 0.14 (0.0086) 0.11, 0.17 <0.001 0.18 - 0.27[0.29 - 0.37] [0.17 - 0.21] [0.066, 0.37]Monsanto Company 09-SY-201UPage 326 of 4711DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval; ND = not detected; NC = not calculated2With 95% confidence, interval contains 99% of the values expressed in the populationof commercial soybean varieties. Negativelimits were set to zero.Table E-17 (continued). Comparison of Fatty Acidsand Vitamin E from MON 87705and Conventional Control(A3525) Soybean RBD Oil Processed from the 2007 U.S. Field ProductionDifference (MON 87705 minusControl)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]RBD Oil Fatty Acid (% TotalFA)22:0 Behenic 0.31 (0.011) 0.35 (0.011) -0.034 (0.0033)-0.044, -0.023 0.0010.30 - 0.50[0.30 - 0.35] [0.33 - 0.38][0.11, 0.70]24:0 Lignoceric 0.14 (0.018) 0.15 (0.018)-0.0056 (0.0036) -0.017, 0.0059 0.2180.088 - 0.23[0.11 - 0.18] [0.11 - 0.19][0, 0.36]Vitamin E (mg/100g FW)11.64 (2.96) 13.09 (2.96) -1.45 (0.67) -3.59, 0.70 0.120 5.36 - 31.55[6.37 - 17.35] [7.72 - 20.35] [0, 56.40]Monsanto Company 09-SY-201UPage 327 of 471Table E-18. Comparison of Amino Acids and Moisture from MON 87705 and Conventional Control (A3525)Soybean Protein Isolate Processed from a 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercialRange[99% Tolerance Int.2]Amino Acid (%DW)Alanine3.73 (0.029)3.71 (0.029)0.017 (0.015)-0.031, 0.0640.3453.62 - 3.90[3.66 - 3.81][3.64 - 3.76][3.39, 4.19]Arginine7.84 (0.094)7.83 (0.094)0.0065 (0.11)-0.34, 0.350.9557.69 - 8.17[7.57 - 8.08][7.68 - 8.04][7.38, 8.51]Aspartic Acid10.88 (0.10)10.87 (0.10)0.013 (0.14)-0.42, 0.450.93010.93 - 11.54[10.58 - 11.19][10.68 - 11.04][10.38, 11.90]Cystine1.21 (0.024)1.18 (0.024)0.035 (0.034)-0.050, 0.120.3511.06 - 1.29[1.17 - 1.28][1.12 - 1.23][0.87, 1.44]Glutamic Acid17.52 (0.23)17.66 (0.23)-0.14 (0.20)-0.77, 0.500.54018.24 - 20.10[16.89 - 18.15][17.25 - 18.02][16.60, 21.24]Glycine3.93 (0.021)3.92 (0.021)0.0027 (0.029)-0.090, 0.0950.9323.86 - 4.04[3.87 - 3.98][3.90 - 3.97][3.71, 4.17]Histidine2.40 (0.019)2.39 (0.019)0.0026 (0.017)-0.052, 0.0580.8882.31 - 2.55[2.35 - 2.44][2.36 - 2.44][2.16, 2.73]Monsanto Company 09-SY-201UPage 328 of 471Table E-18 (continued). Comparison of Amino Acidsand Moisture from MON 87705 and Conventional Control(A3525) Soybean Protein Isolate Processed from a 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Amino Acid (% DW)Isoleucine 4.43 (0.039) 4.47 (0.039) -0.032(0.024) -0.11, 0.046 0.280 4.44 - 4.78[4.35 - 4.48] [4.36 - 4.56] [4.14, 5.10]Leucine 7.33 (0.037) 7.33 (0.037) -0.00005(0.039) -0.12, 0.12 0.999 7.36 - 7.76[7.19 - 7.41] [7.28 - 7.38] [7.10, 8.06]Lysine 5.80 (0.017) 5.76 (0.017) 0.034 (0.015) -0.013, 0.081 0.102 5.81 - 6.03[5.75 - 5.83] [5.74 - 5.81] [5.67, 6.15]Methionine 1.27 (0.024) 1.24 (0.024) 0.035(0.030) -0.061, 0.13 0.327 1.13 - 1.25[1.25 - 1.29] [1.15 - 1.29] [0.99, 1.41]Phenylalanine 5.05 (0.038) 5.07 (0.038) -0.019 (0.046) -0.17, 0.13 0.713 5.04 - 5.50[4.94 - 5.15] [5.01 - 5.14] [4.68, 5.71]Proline 5.02 (0.051) 5.05 (0.051) -0.033(0.049) -0.19, 0.12 0.555 4.54 - 4.99[4.91 - 5.11] [4.92 - 5.17] [4.29, 5.27]Serine 4.96 (0.043) 4.92 (0.043) 0.043 (0.061) -0.11, 0.19 0.513 4.54 - 5.15[4.87 - 5.06] [4.86 - 5.02] [4.00, 5.76]Threonine 3.37 (0.037) 3.35 (0.037) 0.018(0.0085) -0.0086, 0.045 0.118 3.22 - 3.45[3.28 - 3.43] [3.24 - 3.43] [2.99, 3.62]Monsanto Company 09-SY-201UPage 329 of 4711DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the populationof commercial soybean varieties. Negativelimits were set to zero.Table E-18 (continued). Comparison of Amino Acidsand Moisture from MON 87705 and Conventional Control(A3525) Soybean Protein Isolate Processed from a 2007 U.S. Field ProductionDifference (MON 87705 minusControl)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Amino Acid (% DW)Tryptophan1.05 (0.016)1.04 (0.016)0.0071 (0.011)-0.027, 0.0420.5560.90 - 1.06[1.01 - 1.07][0.99 - 1.07][0.76, 1.16]Tyrosine3.46 (0.012)3.45 (0.012)0.0089 (0.013)-0.032, 0.0500.5363.45 - 3.64[3.43 - 3.49][3.43 - 3.48][3.25, 3.85]Valine4.42 (0.052)4.45 (0.052)-0.031 (0.054)-0.20, 0.140.6104.46 - 4.94[4.34 - 4.49][4.29 - 4.58][4.04, 5.27]Moisture (% FW)1.91 (0.28)1.33 (0.28)0.58 (0.27)-0.27, 1.430.1171.32 - 4.71[1.30 - 2.96][0.93 - 1.69][0, 6.54]Monsanto Company 09-SY-201UPage 330 of 4711DW = dry weight; FW = freshweight; S.E. = standard error; CI = confidence interval.2With 95% confidence, interval contains 99% of the values expressed in the populationof commercial soybean varieties. Negativelimits were set to zero.Table E-19. Comparison of Phosphatides from MON 87705 and ConventionalControl (A3525) Soybean LecithinProcessed from a 2007 U.S. Field ProductionDifference (MON 87705 minus Control)AnalyticalComponent1MON 87705Mean ± S.E.1[Range]A3525Mean ± S.E.[Range]Mean ± S.E.[Range]95% CI1(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Int.2]Phosphatides (% FW)L-alpha-Phosphatidic Acid1.10 (0.29)1.24 (0.29)-0.14 (0.41)-1.14, 0.860.7401.30 - 4.19[0.35 - 1.64][0.35 - 1.60][0, 5.64]L-alpha-Phosphatidylcholine4.38 (1.04)7.18 (1.04)-2.80 (1.14)-6.44, 0.830.0912.88 - 7.11[1.93 - 7.85][5.75 - 9.18][0, 11.42]L-alpha-Phosphatidylethanolamine2.45 (0.74)4.99 (0.74)-2.55 (0.97)-5.62, 0.530.0772.21 - 5.50[0.65 - 4.79][4.06 - 6.70][0, 8.78]L-alpha-Phosphatidylinositol2.28 (0.66)4.63 (0.66)-2.36 (0.83)-5.00, 0.290.0662.19 - 5.52[1.13 - 4.53][3.99 - 6.06][0, 8.67]Monsanto Company 09-SY-201U Page 331 of 471 1 Literature range references: a Lundry, et al., 2008. b Padgette et al., 1996.

Table E-20. Literature Ranges for Components in Soybean Meal

Tissue Component Literature Range 1

Proximates (% dw)

Ash 5.2 – 9.1 a Carbohydrates 32.0 – 38.0 b

Fat, total 0.5 -3.30 a Moisture (% fw) 5.58-11.7 a

Protein 47.4 – 59.5 a

Fiber (% dw) a Acid detergent fiber (ADF) 5.2 – 6.7 Neutral detergent fiber (NDF) 7.4 – 12.2

Amino Acids (%dw) a

Alanine 2.18 – 2.59 Arginine 3.29 – 4.49 Aspartic acid 5.18 – 6.83 Cystine/Cysteine 0.6 – 0.92

Glutamic acid 8.05 – 11.21 Glycine 2.02 – 2.40

Histidine 1.32 – 1.63

Isoleucine 2.11 – 2.74

Leucine 3.62 – 4.72 Lysine 2.97 – 3.69

Methionine 0.5 – 0.9 Phenylalanine 2.39 – 3.19

Proline 2.32 – 3.05

Serine 1.97 – 3.3

Threonine 0.80 – 2.24 Tryptophan 0.60 – 2.08 Tyrosine 1.68 – 2.17

Valine 2.29 – 2.92

Anti-Nutrients Trypsin Inhibitors (TIU/mg dw) 3.8 – 17.9 a Phytic Acid (% dw) 1.3 – 4.1 aMonsanto Company 09-SY-201U Page 332 of 471 Table E-21. Literature Ranges for Components in Soybean Protein

Isolate

Tissue Component Literature Range or Value 1

Proximates Moisture (% fw) 3.9 – 7.0

Amino Acids (%dw)

Alanine NA Arginine 6.67 Aspartic acid NA Cystine/Cysteine 1.05

Glutamic acid NA Glycine NA

Histidine 2.3

Isoleucine 4.25

Leucine 6.78 Lysine 5.33

Methionine 1.13 Phenylalanine 4.59

Proline NA

Serine NA

Threonine 3.14 Tryptophan 1.12 Tyrosine NA

Valine 4.1

1 Literature range or value reference: Lundry, et. al., 2008.

Table E-22. Literature Ranges for Components in Soybean Lecithin

Tissue Component Literature Range 1

Phosphatides (%fw)

α -Phosphatidic Acid 0.2 – 14.0

α -Phosphatidylcholine 12.0 – 46.0

α -Phosphatidylethanolamine 8.0 – 34.0

α -Phosphatidylinositol 1.7 – 21.0

Literature range reference: 1 Lundry, et. al., 2008.Monsanto Company 09-SY-201U Page 333 of 471 Table E-23. Literature Ranges for Components in Soybean Oil

Tissue Component Literature Range 1

Fatty Acids (FA) 14:0 Myristic ND – 0.2 a

16:0 Palmitic 7 - 12 b

16:1 Palmitoleic ≤ 0.2 b
17:0 Margaric [Heptadecanoic] ND – 0.1 a 17:1 9c Heptadecenoic ND – 0.1 a

18:0 Stearic 2 – 5 b

18:1 Oleic 19 – 34 b

18:2 Linoleic 48 – 60 b

18:3 Linolenic 2 -10 b

20:0 Arachidic 0.1 – 0.6 a

20:1 Eicosenoic ND – 0.5 a

20:2 Eicosadienoic ND – 0.1 a

22:0 Behenic ND – 0.7 a

Vitamins mg/100g fw

Vitamin E 0.9 – 35.2 a

1 Literature range references: a Codex, 2005 (% Total FA). b Lundry, et al., 2008, (% FW). ND = not detected.Monsanto Company 09-SY-201U Page 334 of 471 Appendix E References

Chardigny J.M., J.L. Sébédio, and O. Berde ux. 1996. Trans Polyunsaturated Fatty Acids:

Occurrence and Nutritional Implications. Ann. Appl. Lipid Reearch 2:1-33.

Codex. 2005. Codex Standard for named ve getable oils. Codex Alimentarus.

George, C., W.P. Ridley, J.C. Obert, M. A. Nemeth, M.L. Breeze, and J.D. Astwood. 2004. Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn (Zea mays L.). Jour nal of Agricultural and Food Chemistry 52:4149-4158.

Ledoux, M., P. Juaneda, and J-L Sebedio. 2007. Tran fatty acids: Definition and occurrence in foods. European Journa l of Lipid Science Technology 109:891-900.

Lundry, D.R., Ridley, W. P., Meyer, J.J., Ri ordan, S.G., Nemeth, M.A., Trujillo, W.A., Breeze, M.L., and Sorbet, R. 2008. Compos ition of Grain, Forage, and Processed Fractions from Second-generation Glyphos ate-Tolerant Soybean, MON 89788, Is Equivalent to That of Conventional Soybean (Glycine max L.). J. Agric. Food Chem.,

56:4611-4622.

NOPA. 2006. Trading rules for the purchase an d sale of soybean meal. National Oilseed

Processors Associati on, Washington, D.C.

OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean: key food and feed nutrients and anti-nutrients. OECD ENV/JM/MONO(2001)15.

Padgette, S.R., N.B. Taylor, D.L. Nida, M.R. Bailey, J. MacDonald, L.R. Holden, and R.L. Fuchs. 1996. The composition of glyphosate- tolerant soybean seeds is equivalent to that of conventional soybeans. Journal of Nutrition 126:702-716.

Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. Comparison of the nutritional profile of glyphos ate-tolerant corn event NK603 with that of conventional corn (Zea mays L.). J ournal of Agricultural and Food Chemistry

50:7235-7243.

Wolff, R.L. 1993. Further studies on artificial geomet rical isomers of α -linolenic acid in edible linolenic acid-containing oils. Jour nal fo the American Oil Chemists’ Society

3:219-224.Monsanto Company 09-SY-201U Page 335 of 471 Appendix F. Materials and Methods for Seed Dormancy and Germination Analyses of MON 87705

F.1 Materials

MON 87705, a conventional soybean cont rol (A3525), and commercial soybean reference variety starting s eed were produced in Jefferson County, IA; Boone County, IN; and Macon County, MO in 2007.

F.2 Characterization of the Materials

For the MON 87705, conventional soybean cont rol, and commercial soybean reference variety starting seed, the pr esence or absence of MON 87705 was verified by eventspecific polymerase chain reaction (PCR) an alyses. The results of these analyses confirmed the presence of MON 87705 in th e MON 87705 starting seed and the absence

of MON 87705 in the control and reference variety seed.

F.3 Performing Facility and Experimental Methods

Dormancy and germination evaluations were conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigator wa s qualified to conduct seed dormancy and germination testing consistent with the standards established by the Association of Official Seed Analysts, a seed trade association (AOS A, 2000; AOSA, 2006; AOSA, 2007).

Six germination chambers were used in the evaluation and each chamber was maintained dark under one of the following six temperat ure regimes: consta nt temperature of approximately 10, 20 or 30 o C or alternating temperatures of approximately 10/20, 10/30,

or 20/30 o C. The alternating temperature regi mes were maintained at the lower temperature for 16 hours and the higher temper ature for eight hours. The temperature inside each germination chamber was m onitored and recorded every 15 minutes throughout the duration of the study.

Germination towels for MON 87705, control, an d reference materials were prepared per facility SOPs. Each germination towel represented one replication. The types of data collected depended on the temperature regime. Each rolled germin ation towel in the AOSA-recommended temperatur e regime (i.e., 20/30 o C) was assessed periodically during the study for normal germinated, a bnormal germinated, hard (viable and nonviable), dead, and firm swollen (viabl e and nonviable) seed as defined by AOSA guidelines (AOSA, 2006; AOSA, 2007). Each rolled germination towel in the additional temperature regimes (i.e., 10, 20, 30, 10/20 and 10/30 o C) was assessed periodically during the study for germinated, hard (viabl e and nonviable), dead, and firm swollen (viable and nonviable) seed.Monsanto Company 09-SY-201U Page 336 of 471 F.4 Statistical Analysis

Statistical analyses were performed by th e Monsanto Statistics Technology Center. Analysis of variance was conducted accord ing to three randomized complete block designs with four replications, with the exception that the germination towels were arbitrarily arranged in each bucket and not necessarily randomized. SAS was used to compare MON 87705 to the conventional soybean control within ea ch seed production site for the following germination characteris tics: percent germinated (categorized as percent normal germinated and percent abnormal germinated for the AOSA temperature regime), percent viable hard seed, percent dead, and percent viable firm-swollen seed. The level of statistical signifi cance was predetermined to be α = 0.05. The MON 87705 substance was not statistically compared to the reference substances, nor were comparisons made across temperature regimes. The minimum and maximum mean values (reference range) were determined from the reference substances at each site.

F.5 Individual Site Seed Dormancy and Germination Analysis

There were no statistically significant differences dete cted between MON 87705 and the control for any of the measured characteristic s (i.e., percent germinated, viable hard, or viable firm swollen seed) in any temperatur e regime for seed produced at the IN site. Seven statistically si gnificant differences were de tected between MON 87705 and the control (Table F-2) for seed produced at the IA and MO sites. MON 87705 had lower percent normal germination than the control at 20/30 oC for seed produced at the IA site (89.3 vs. 94.0%). At 30 ° C, MON 87705 had lower percent germination than the control for seed produced at the IA site (82.8 vs. 95.5%) and higher percent germination than the control for seed produced at the MO site (100.0 vs. 96.3%). MON 87705 had lower percent viable hard seed than the control at 20 ° C for seed produced at the MO site (0.0 vs. 0.8%). Percent dead s eed was higher for MON 87705 than the control at 30 oC (17.3 vs. 4.5%) and 20/30 oC (6.0 vs. 2.5%) for seed produced at the IA site and lower for

MON 87705 than the control at 30 oC for seed produced at th e MO site (0.0 vs. 3.8%).Monsanto Company 09-SY-201UPage 337 of 471Table F-1. Starting Seed of MON 87705,Control and Commercial Reference Soybean Varieties Used in DormancyAssessmentProduction SiteSubstance NameSubstance TypePhenotype1Sample ID NumberIAMON 87705TestModified oil profileRPN07273-001IAA3525ControlConventionalRPN07273-002IAHoegemeyer 333ReferenceConventionalRPN07273-005IAStewart 3454ReferenceConventionalRPN07273-006IAStine 3600-0ReferenceConventionalRPN07273-007IALewis 372ReferenceConventionalRPN07273-008INMON 87705TestModified oil profileRPN07273-009INA3525ControlConventionalRPN07273-010INMidland 363ReferenceConventionalRPN07273-013INNK S33A8ReferenceGlyphosate tolerant2RPN07273-014INAsgrow AG3505ReferenceGlyphosate tolerant2RPN07273-015INFS 3591ReferenceConventionalRPN07273-016MOMON 87705TestModified oil profileRPN07273-017MOA3525ControlConventionalRPN07273-018MOHoegemeyer 333ReferenceConventionalRPN07273-021MOStewart 3454ReferenceConventionalRPN07273-022MOStine 3600-0ReferenceConventionalRPN07273-023MOLewis 372ReferenceConventionalRPN07273-0241MON 87705 possesses the improved fatty acid profile trait; the control and reference soybean varieties do not possess the trait.2Glyphosate tolerant = Commercially available Roundup Ready soybean 40-3-2 variety.Monsanto Company 09-SY-201UPage 338 of 471Table F-2. Comparison of MON 87705 to the Control for Dormancy and Germination CharacteristicsTemp.RegimeCategoryIA1Mean % (S.E.)2IN1Mean % (S.E.)2MO1Mean % (S.E.)2MON 87705 ControlReferenceRange3MON 87705 ControlReferenceRange3MON 87705 ControlReferenceRange310 °C Germinated96.8 (1.5) 98.0 (1.2) 99.3 – 100.097.5 (1.7) 98.8 (0.5) 98.5 – 99.398.8 (0.6) 98.5 (0.6) 96.5 – 99.3Viable Hard0.0 (0.0) 0.3 (0.3) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.3 (0.3) 0.5 (0.3) 0.5 – 1.5Dead3.0 (1.6) 1.8 (1.0) 0.0 – 0.8 2.3 (1.7) 0.8 (0.3) 0.8 – 1.3 1.0 (0.7) 0.8 (0.5) 0.3 – 1.8Viable FirmSwollen0.3 (0.3) 0.0 (0.0) 0.0 – 0.0 0.3 (0.3) 0.5 (0.3) 0.0 – 0.5 0.0 (0.0) 0.3 (0.3) 0.0 – 0.320 °C Germinated97.5 (1.3) 98.5 (0.6) 98.8 – 99.5 99.8 (0.3) 99.8 (0.3) 99.5 – 99.8 100.0 (0.0) 98.3 (0.5) 95.0 – 98.8Viable Hard0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.3 (0.3) 0.0 – 0.0 0.0 (0.0) * 0.8 (0.3) 0.0 – 0.5Dead2.5 (1.3) 1.5 (0.6) 0.5 – 1.3 0.3 (0.3) 0.0 (0.0) 0.3 – 0.5 0.0 (0.0) 1.0 (0.6) 0.8 – 5.0Viable FirmSwollen0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.030 °CGerminated82.8 (3.1)* 95.5 (1.3) 97.5 – 98.595.5 (1.2) 94.8 (0.9) 96.0 – 99.5 100.0 (0.0) * 96.3 (0.9) 91.0 – 98.5Viable Hard0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.0 (0.0) 0.0 (0.0) 0.0 – 0.5Dead17.3 (3.1) * 4.5 (1.3) 1.5 – 2.5 4.5 (1.2) 5.3 (0.9) 0.3 – 4.0 0.0 (0.0) * 3.8 (0.9) 1.3 – 8.5Viable FirmSwollen0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0Monsanto Company 09-SY-201UPage 339 of 471Table F-2 (cont.). Comparison of MON 87705 to theControl for Dormancy and Germination CharacteristicsTemp.RegimeCategoryIA1Mean % (S.E.)2IN1Mean % (S.E.)2MO1Mean % (S.E.)2MON 87705 ControlReferenceRange3MON 87705 ControlReferenceRange3MON 87705 ControlReferenceRange210/20°C Germinated99.3 (0.3) 99.5 (0.5) 98.8 – 99.599.5 (0.3) 99.8 (0.3) 99.3 – 100.099.3 (0.5) 99.5 (0.5) 97.8 – 99.8Viable Hard0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.3 (0.3) 0.3 (0.3) 0.0 – 0.5Dead0.8 (0.3) 0.5 (0.5) 0.5 – 1.3 0.5 (0.3) 0.3 (0.3) 0.0 – 0.5 0.5 (0.3) 0.3 (0.3) 0.0 – 2.3Viable FirmSwollen0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.010/30°C Germinated95.3 (1.3)97.5 (1.0)99.3 – 99.5 99.3 (0.5) 99.3 (0.3) 98.8 –99.5 99.5 (0.3) 99.5 (0.3) 96.8 – 99.5Viable Hard0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.0 (0.0) 0.0 (0.0) 0.0 – 0.5Dead4.8 (1.3) 2.5 (1.0) 0.5 – 0.8 0.8 (0.5) 0.8 (0.3) 0.5 – 1.3 0.5 (0.3) 0.5 (0.3) 0.5 – 2.8Viable FirmSwollen0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.020/30°CNormalGerminated89.3 (1.3) * 94.0 (1.1) 95.8 – 97.396.0 (0.8) 94.5 (1.0) 57.0 – 98.592.0 (0.9) 92.3 (1.3) 81.0 – 95.0(AOSA)AbnormalGerminated4.8 (1.3) 3.5 (1.4) 2.0 – 3.8 3.5 (1.0) 5.5 (1.0) 1.3 – 42.5 6.5 (0.6) 6.0 (1.1) 3.8 – 14.8Viable Hard0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.0 (0.0) 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.5 (0.3) 0.0 – 0.5Dead6.0 (0.4) * 2.5 (0.5) 0.0 – 1.0 0.5 (0.3) 0.0 (0.0) 0.3 – 1.0 1.5 (0.5) 1.3 (0.3) 0.5 – 4.3Viable FirmSwollen0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05).† No statistical comparison could be made due to lack of variability in the data.1Site codes are as follows: IA = Jefferson County, IA; IN = Boone County, IN; MO = Macon County, MO.2Means based on four replicates (n = 4) of approximately 100 seeds. In some instances, the total percentage of both MON 87705and the control did not equalexactly 100% due to numerical rounding of the means. S.E. = Standard Error.3Minimum and maximum mean values determined from commercially available reference soybean varietiesMonsanto Company 09-SY-201U Page 340 of 471 Appendix F References

AOSA. 2000. Tetrazolium Testing Handbook -Contribution No. 29 to the Handbook on Seed Testing. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2006. Seedling Eval uation Handbook, Contribution No. 35 to the Handbook on Seed Testing. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2007. Rules for Testing Seeds. Associa tion of Official Seed Analysts, Lincoln,

Nebraska.Monsanto Company 09-SY-201U Page 341 of 471 Appendix G. Material, Methods and Indi vidual Site Results from Phenotypic, Agronomic and Ecological Intera ctions Analyses of MON 87705

G.1 Materials

The materials for phenotypic assessments include: MON 87705, a conventional soybean control (A3525), and 17 commercially available soybean varieties as references. The

references contain both conventional s oybean and Roundup Ready soybean 40-3-2 varieties. The list of soybean varieties planted in each site is presented in Table G-1. The identities of MON 87705 and control (A352 5) seed were confirmed by PCR analysis prior to use.

G.2 Field Sites and Plot Design

Data were collected from fi eld trials conducted in 2007 at 17 sites within U.S. soybean production regions (Section VIII, Table VIII-3). The 17 sites provided a range of environmental and agronomic conditions repres entative of major U. S. soybean-growing regions. The field cooperators at each site were familiar with the growth, production, and evaluation of soybean characteristics.

The experiment was established at each of the 17 sites in a randomized complete block design with three replications. Each plot at the IA1, IL2, IN 1, and MO2 sites consisted of eight 30 ft long rows with in ter-row spacing of approximately 30 inches. Rows # 2 and 3 were designated for the collection of phenotypi c, abiotic stress response, disease damage, and arthropod damage data. Rows # 5–7 were designated for the co llection of arthropod samples. Rows # 1, 4, and 8 were used as buffer rows. Each plot was surrounded by an approximately 10 ft, four-row border of a commercially available soybean variety to create a continuous soybean stand across the pl ot area to ensure coll ection of more robust arthropod abundance data with in the test area.

Each plot at the AR, IA2, IL 1, IL3, IN2, KS, MI, MO3, MO4, NE, OH, PA, and WI sites consisted of four 20 ft long rows with in ter-row spacing of approximately 30 inches. Rows # 2 and 3 were designated for the coll ection of phenotypic, ab iotic stress response, disease damage, and arthropod da mage data. Rows # 1 and 4 were used as buffer rows. The entire plot area was surrounded by an approximately 10 ft, four-row border of a commercially available soybean variety.Monsanto Company 09-SY-201UPage 342 of 471Table G-1. Starting Seedfor Phenotypic AssessmentsSubstanceSubstance typeRelativeMaturityGroupPhenotype1Monsanto Lot #Sites2MON 87705Test3.5Improved Fatty AcidProfileGLP-0702-18254-SAll sitesA3525Control3.5ConventionalGLP-0702-18252-SAll sitesA3244Reference3.2ConventionalGLP-0703-18458-SAll sitesCroplan HT3596STSReference3.5ConventionalGLP-0703-18386-SAR, IL2, KS, MO3, PAPioneer 93M50Reference3.5Roundup Ready3GLP-0604-17240-SAR, IL2, KS, MO3, PAChannel Bio 3461Reference3.4ConventionalGLP-0605-17332-SAR, IL2, KS, MO3, PAPioneer 93M52Reference3.5ConventionalGLP-0703-18407-SAR, IL2, KS, MO3, PAHoegemeyer 333Reference3.2ConventionalGLP-0703-18383-SIA1, IL3, MI, MO4, WIStewart 3454Reference3.4ConventionalGLP-0703-18426-SIA1, IL3, MI, MO4, WIStine 3600-0Reference3.6ConventionalGLP-0605-17336-SIA1, IL3, MI, MO4, WILewis 372Reference3.7ConventionalGLP-0604-17261-SIA1, IL3, MI, MO4, WIMidland 363Reference3.3ConventionalGLP-0703-18384-SIA2, IN1, NENK S33A8Reference3.3Roundup Ready3GLP-0604-17243-SIA2, IN1, NEAsgrow AG3505Reference3.5Roundup Ready3GLP-0604-17250-SIA2, IN1, NEFS 3591Reference3.5ConventionalGLP-0703-18387-SIA2, IN1, NEStine 3300-0Reference3.3ConventionalGLP-0605-17335-SIL1, IN2, MO2, OHDeKalb DKB34-51Reference3.4Roundup Ready3GLP-0604-17254-SIL1, IN2, MO2, OHGarst 3585NReference3.5ConventionalGLP-0703-18388-SIL1, IN2, MO2, OHCrows C37003NReference3.7ConventionalGLP-0703-18390-SIL1, IN2, MO2, OH1MON 87705 expresses the improved fatty acid profile trait; whereas the conventional soybean control and reference varieties donot express the improved fattyacid profile trait.2MON 87705, the control, and reference material A3244 were planted at all field sites; the remaining reference varieties were site-specific. Site codes are asfollows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, IL; IL2 = Stark County, IL, IL3 = WarrenCounty, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS =Pawnee County, KS; MI = Ottawa County, MI; MO2 = Lincoln County, MO, MO3 = St.Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH= Fayette County, OH; PA = Berks County, PA; WI = Walworth County, WI.3Commercially available glyphosate-tolerant (Roundup Ready 40-3-2) soybean variety.Monsanto Company 09-SY-201UPage 343 of 471Table G-2. Field and Planting InformationSite1Plantingdate(mm/dd/yr)Seedingrate(seeds/ft)2Plantingdepth(in)Plotsize(ft)3Rows/plotSoil series, organic matter, pHCropping history20062005AR05/26/200780.7510 x 204Crowley silt loam, 2.5%, 6.2RiceSoybeanIA105/23/200781.2520 x 308Tainter/Mahaska silty clay loam, 4.02%, 6.75CornCornIA205/26/200781.510 x 204Tama-Muscatine silty clay loam, 2.8%, 6.3Milk thistleCornIL106/04/200781.010 x 204Silt loam, 2.65%, 6.59MiloFallowIL205/31/200781.520 x 308Plano silt loam, 3.5%, 6.4CornSoybeanand CornIL305/23/200781.010 x 204Sable silty clay loam, 6.0%, 5.6-7.3CornSoybeanIN105/30/200781.520 x 308Crosby silty clay loam, 2.4%, 6.8CornSoybeanIN206/08/200781.2510 x 204Reesville silt loam, 1.4%, 5.8CornCornKS05/22/200791.010 x 204Farnum loam, 2.6%, 7.6WheatAlfalfaMI05/23/200781.510 x 204Nester loam, 2.1%, 6.5CornSoybeanMO205/30/200781.520 x 308Crider silt loam, 2.3%, 6.7CornSoybeanMO305/25/200781.510 x 204Kennebec silt loam, 2.6%, 6.3CornSoybeanMO406/07/200781.010 x 204Gorin silt loam, 4.2%, 5.8FescueFescueNE05/21/200781.010 x 204Hastings silt loam, 3.0%, 6.6SoybeanSoybeanOH05/22/200781.010 x 204Crosby silt loam, 1.8%, 6.6CornSoybeanPA06/09/200781.510 x 204Philo/Atkins silt loam, 2.4%, 5.8TomatoCornWI05/29/200781.010 x 204Radford silt loam, not available, not availableWheatSoybean1Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County,IL; IL2 = Stark County, IL,IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS;MI = Ottawa County, MI; MO2 =Lincoln County, MO,MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA; WI =WalworthCounty, WI.2The KS site planted the production at nine seeds per foot as specified in the study protocol.3Width × length.Monsanto Company 09-SY-201U Page 344 of 471 G.3 Planting and Field Operations

Planting information is listed in Table G-2. Agronomic practices used to prepare and maintain each study site were characteristic of those used in each respective geographic region. Herbicides containing glyphosate we re not used to avoid injury to the conventional soybean control or conventional soybean referen ce varieties and to ensure all plants were managed uniformly.

G.4 Phenotypic Observations

The description of the charac teristics measured and the de signated developmental stages

where observations occurred are listed in Section VIII, Table VIII-1.

G.5 Ecological Observations

Ecological interactions (i.e., interactions between the crop plants and their receiving environment) were used to characteri ze MON 87705 by evaluating plant response to abiotic stressors, disease damage, arthropod damage, and pest and beneficial arthropod abundance in the plots us ing the following methods:

G.6 Abiotic Stress Response, Disease Damage, and Arthropod Damage

MON 87705 and the conventional soybean contro l were evaluated at each of 17 sites (AR, IA1, IA2, IL1, IL2, IL3, IN1, IN2, KS, MI, MO2, MO3, MO4, NE, OH, PA, and WI) for differences in plant response to abiotic stressors, disease damage, and arthropod damage. Three abiotic stresso rs, three diseases, and three arthropod pests were evaluated four times during the growing seas on at the following intervals:

Observation 1: V2 – V4 growth stage Observation 2: R1 – R2 growth stage Observation 3: R3 – R5 growth stage Observation 4: R6 – R8 growth stage

The principal investigator at each site c hose abiotic stressors, diseases, and arthropod pests that were either activel y causing plant injury in the study area or were likely to occur in soybean during a gi ven observation period. Th erefore, abiotic stressors, diseases, and arthropod pests assessed often varied between observations at a site and

between sites.

Abiotic stressors and disease damage were assessed in Rows # 2 and 3 of each plot using a continuous 0 – 9 rating scale of increasin g symptomology. Data were collected numerically and then placed into one of the following categ ories for reporting purposes:

Rating Severity of plant damage 0 none (no symptoms observed) 1 – 3 slight (symptoms not da maging to plant development) 4 – 6 moderate (intermediate between slight and severe) 7 – 9 severe (symptoms damaging to plant development)Monsanto Company 09-SY-201U Page 345 of 471 Arthropod damage was assessed in Rows # 2 an d 3 of each plot on the upper four nodes of 10 non-systematically selected plants usi ng arthropod-specific 0 – 5 rating scales of increasing symptomology listed be low. Data were collected numerically and then placed into one of the categories in the follow ing rating scales fo r reporting purposes:

Defoliating arthropods (e.g., corn earworm, bean leaf beetle, Japanese beetle, soybean looper) Rating Defoliation (%) Severity of plant damage 0 none none (no symptoms observed) 1 1 – 20 % slight (symptoms not damaging to plant development)

2 21 – 40% moderate (intermediate between slight and severe) 3 41 – 60%

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80%

Pod feeding arthropods (e.g., corn earworm, bean leaf beetle, stink bug, Lygus bug on reproductive plant parts) Rating Damaged pods (%) Severity of plant damage 0 none none (no symptoms observed)

1 1 – 20 % slight (symptoms not damaging to plant development)

2 21 – 40% moderate (intermediate between slight and severe) 3 41 – 60%

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80%

Leafhoppers (e.g., potato leafhopper) Rating Foliar damage (%) Severity of plant damage 0 none none (no symptoms observed)

1 1 – 50% of foliage with leaf yellowing; no leaf puckering or leaf margin necrosis slight (symptoms not damaging to plant development)

2 1 – 50% of foliage with leaf yellowing, leaf puckering and/or le af margin necrosis moderate (intermediate between slight and severe) 3 > 50% of foliage with leaf yellowing; no leaf puckering or leaf margin necrosis

4 > 50% of foliage with leaf yellowing, leaf puckering, and/or leaf margin necrosis severe (symptoms damaging to plant development) 5 > 50% of foliage with necrotic leaves (leaves dead due to leafhopper damage)Monsanto Company 09-SY-201U Page 346 of 471 Aphids (e.g., soybean aphid) Rating Aphids present Severity of plant damage 0 none none (no symptoms observed)

1 1 – 100 aphids per plant; no leaf puckering slight (symptoms not damaging to plant development)

2 101 – 250 aphids per plant; no leaf puckering moderate (intermediate between slight and severe) 3 ≥ 250 aphids per plant with leaf puckering

4 ≥ 250 aphids per plant with leaf puckering and leaf yellowing and/or necrosis severe (symptoms damaging to plant development)

5 ≥ 250 aphids per plant with plant stunting

For each abiotic stress response, disease dama ge, and arthropod damage observation at a site, the range of injury severity ratings obs erved across all three re plications for each of MON 87705, the conventional soybean control and reference soyb ean varieties at the site was determined, and the numeric ranges were then converted to categorical ranges (e.g., none, slight, moderate, severe ) for reporting purposes.

G.7 Arthropod Abundance

Pest and beneficial arthropods were collected at th e IA1, IL2, IN1, and MO2 sites three times during the growing season at the following intervals:

Collection 1: R1 – R2 growth stage

Collection 2: R3 – R5 growth stage

Collection 3: R6 – R8 growth stage

Arthropods were collected using a beat shee t sampling method (Kogan and Pitre, 1980). The beat sheet was an approximately 36 × 42 inch white, vinyl sheet spread between the plants of two adjacent rows. Plants were sh aken vigorously along the length of each side of the beat sheet to dislodge arthropods from the plants. A total of four subsamples were collected in this way from each plot. Speci fically, two subsamples were collected from Rows # 5 and 6 of each plot (subsamples 1 and 3) and two subsamples were collected from Rows # 6 and 7 of each plot (subsamples 2 and 4). The subsamples collected from the same row were at least 10 ft apart and at least 3 ft from the edge of each plot. The four subsamples were combined into one pr e-labelled container and placed on dry ice. The samples were then sent overnight to the Monsanto Regul atory Environmental Science Center for arthropod id entification and enumeration.

A maximum of the six most abundant pest an d six most abundant be neficial arthropods

were determined for each collection interval from each individual si te. These specific arthropods were then enumerated across all sa mples from a given collection interval at each individual site. The arthropods assesse d often varied between collection intervals

from a site and between sites due to differe nces in temporal ac tivity and geographical

distribution of the taxa.Monsanto Company 09-SY-201U Page 347 of 471 G.8 Ecological Interact ions Evaluation Criteria

For the assessments of abiotic stress res ponse, disease damage, and arthropod damage, MON 87705 and the conventional soybean c ontrol were considered different in susceptibility or toleran ce to an abiotic st ressor, disease, or arthr opod pest on a particular observation date if the range of injury severity to MON 87705 did not overlap with the range of injury severity to the control ac ross all three replications. These data are categorical and, therefore, were not subjected to statistical analysis. For each observation at a site, the range of injury severity acros s the commercially available reference soybean varieties provided data that are representativ e of commercial soybean varieties. Pest and beneficial arthropod abundance data were qua ntitatively evaluate d and subjected to statistical analysis, as appropriate.

G.9 Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summarization, and analysis. Personnel assessed that measurements were taken properly, data were consistent with expectations based on experience with the cr op, and the experiment was carefully monitored. Prior to analysis, the overall dataset was evaluated fo r evidence of biologically relevant changes and for possible evidence of an unexpected plant response. Any unexpe cted observations or issues that would impact the evaluati on objectives were noted. Data were then subjected to statistical analys is as indicated below.

G.10 Statistical Analysis

Analysis of variance was conducted according to a randomized complete block design using SAS (Version 9.2). The level of significance was α = 0.05. MON 87705 was compared to the control substance within each site (individual-site analysis) and pooled across all sites (combined-site analysis) for early stand count, seedling vigor, days to 50% flowering, plant height , lodging, shattering, final stand count, seed moisture, 100 seed weight, seed test weight, and yi eld. Growth stage, flower color, plant pubescence, abiotic stress response, disease damage, and arthro pod damage data were not statistically analyzed. Arthropod pest a bundance and beneficial arth ropod abundance data were statistically analyzed only within individua l collection intervals and sites due to the variation in temporal activity and geog raphical distribution of the taxa.

No statistical comparisons were made between MON 87705 and reference soybean varieties. The reference range for each measured phenotypic characteristic was

determined from the minimum and maximu m mean values collected from the 17 reference soybean varieties pl anted among the sites. The re ference range for the plant response to abiotic stressor, disease, arth ropod damage, and abundance of each arthropod evaluated from a given collect ion and site was determined from the minimum and

maximum mean values collected from the refere nce varieties at the site. Thus, reference ranges for abiotic stressor, disease susceptibility, arthropod damage, and arthropod abundance were specific to each collect ion/observation interval and site.

G.11 Individual Field Site Plant Growth and Development Results and DiscussionMonsanto Company 09-SY-201U Page 348 of 471 In the individual-site analysis , lack of variability in the data precluded statistical comparisons between MON 87705 and the contro l for seedling vigor at the IL1, IL3, MO4, and PA sites; days to 50% flowering at the IA2, IN 1, NE, and WI sites; plant lodging ratings at the IL1, IN1, MO2, MO3, and NE sites; pod shatteri ng at the IL1, IL2, IL3, IN2, MI, MO2, MO3, NE, OH, PA, and WI si tes; and test weight at the WI site. In each of these cases, however, the mean for MON 87705 and the mean for the control were the same value or nearly the same value (Table G-3).

In the individual-site analysis, a total of 30 statistically significant differences were detected out of 161 comparisons between MON 87705 and the conventional soybean control (Table G-3). These differences were distributed among nine of the 13 phenotypic characteristics. Early stand count was lowe r for MON 87705 than the control at the AR site (101.7 vs. 155.3 plants/plot) , the IL1 site (253.3 vs. 271. 0 plants/plot), the IL2 site (240.6 vs. 284.6 plants/plot), the OH site (112. 0 vs. 159.0 plants/plot), and the PA site (262.3 vs. 288.0 plants/plot). MON 87705 flowered later than the control at the IL2 site (208.0 vs. 205.3 days after 1 Jan. 2007), the IN 2 site (204.7 vs. 203.0 days after 1 Jan. 2007), the MO2 site (193.7 vs. 191.0 days after 1 Jan. 2007), the MO4 site (204.3 vs. 201.3 days after 1 Jan. 2007), and the PA site (209.0 vs. 207.3 days after 1 Jan. 2007).

Plants of MON 87705 were taller than the cont rol at the IN2 site (35.4 vs. 33.6 inches) but shorter than the control at the IA1 (40.3 vs. 42.9 inches), the MO3 (29.6 vs. 32.0 inches), and the PA (28.6 vs. 32.0 inches) sites. MON 87705 had more lodging than the

control at the IN2 s ite (4.0 vs. 1.7 rating). Final stand count was lower for MON 87705 than the control at the AR (98.0 vs. 153. 7 plants/plot), the IA1 (172.8 vs. 213.0 plants/plot), the IL1 (254.3 vs. 276.0 plants/plot) , the IL2 (206.6 vs. 248.1 plants/plot), the IN1 (226.3 vs. 255.7 plants/plot), and the OH (116.7 vs. 156.7 plants/plot) sites. Seed

moisture was lower for MON 87705 than the co ntrol at the KS (11. 8 vs. 13.4%) and the MO3 (12.0 vs. 14.4%) sites. The weight of 100 seeds was lower for MON 87705 than the control at the IA2 (13.8 vs. 14.4 g), MI (19.6 vs. 21.3 g), and MO4 (13.3 vs. 14.5 g) sites. Test weight was great er for MON 87705 than the control at the IN1 site (50.1 vs. 42.1 lb/bu), but less than the control at the MO4 site (50.7 vs. 53.8 lb/bu). Yield was

lower for MON 87705 than the control at the IL 2 (43.5 vs. 51.2 bu/ac) and MO3 (21.7 vs. 31.8 bu/ac) sites. Considering that the statistical differen ces for plant height, lodging, seed moisture, test weight, and yield that we re detected in the individual-site analyses

were not detected in the combined site anal ysis, this suggests thes e differences were not indicative of a consistent plan t response associated with the trait and are unlikely to be biologically meaningful in terms of increased weed potential of MON 87705 compared to the conventional soybean control. While some statistical differences were detected in the combined-site analysis, the assessed phenotypic values of MON 87705 were within the range of values expected for commercial soybean.Monsanto Company 09-SY-201UPage 349 of 471Table G-3. Phenotypic Comparison of MON 87705 to the Conventional Soybean Control within Each SitePhenotypic Characteristic (units)Early stand count (#/plot)Seedling vigor(1-9 scale)Days to 50% flowering Flower color2Plant pubescence2Site1MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.) MON 87705 Control MON 87705 ControlAR 101.7* (13.9) 155.3 (17.5)6.0 (0.6) 5.3 (0.3)180.0 (1.2) 178.0 (1.2)Purple Purple Hairy HairyIA1 261.7 (8.9) 282.6 (5.4)3.0 (0.6) 2.7 (0.7)192.7 (0.3) 191.7 (0.3)Purple Purple Hairy HairyIA2 266.7 (5.6) 282.3 (2.3)3.3 (0.3) 3.0 (0.0)197.0‡ (0.0) 197.0 (0.0)Purple Purple N/A N/AIL1 253.3* (2.7) 271.0 (5.1)1.0‡ (0.0) 1.0 (0.0)201.0 (0.0) 201.3 (0.3)Purple Purple Hairy HairyIL2 240.6* (8.5) 284.6 (3.5)1.0 (0.0) 1.0 (0.0)208.0* (1.0) 205.3 (0.3)Purple Purple Hairy HairyIL3 283.3 (11.0) 302.0 (9.9)2.0‡ (0.0) 2.0 (0.0)190.0 (1.0) 188.3 (0.7)Purple Purple Hairy HairyIN1 222.8 (15.1) 248.6 (5.8)2.3 (0.3) 3.0 (1.0)207.0‡ (0.0) 207.0 (0.0)Purple Purple Hairy HairyIN2 257.3 (6.9) 259.7 (7.2)3.7 (0.3) 3.3 (0.7)204.7* (0.3) 203.0 (0.0)Purple Purple Hairy HairyKS 158.3 (15.9) 180.7 (8.7)4.0 (0.0) 4.0 (0.0)191.0 (0.6) 191.0 (0.0)Purple Purple Hairy HairyMI 320.3 (1.5) 303.7 (14.3)3.3 (0.3) 4.0 (0.6)198.7 (0.7) 198.0 (0.0)Purple Purple Hairy HairyMO2 288.6 (8.0) 299.5 (6.7)5.3 (0.3) 5.0 (0.0)193.7* (0.3) 191.0 (0.6)Purple Purple Hairy HairyMO3 237.3 (17.5) 280.0 (5.8)4.7 (0.7) 3.3 (0.3)191.7 (1.3) 190.0 (1.5)Purple Purple Hairy HairyMO4 201.3 (12.8) 218.3 (6.6)2.0‡ (0.0) 2.0 (0.0)204.3* (0.7) 201.3 (0.3)Purple Purple N/A N/ANE 308.0 (6.1) 289.3 (13.1)3.0 (0.0) 3.0 (0.0)192.0‡ (0.0) 192.0 (0.0)Purple Purple Hairy HairyOH 112.0* (11.0) 159.0 (10.2)3.7 (0.9) 3.3 (0.7)199.3 (0.7) 197.3 (0.9)Purple Purple Hairy HairyPA 262.3* (6.6) 288.0 (4.2)2.0‡ (0.0) 2.0 (0.0)209.0* (0.0) 207.3 (0.3)Purple Purple Hairy HairyWI 221.0 (20.0) 249.7 (2.9)1.3 (0.3) 1.7 (0.7)207.0‡ (0.0) 207.0 (0.0)Purple Purple Hairy Hairy* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05).‡ Indicates a lack of variability in the data which precluded statistical analysis.N/A = Data not available or excluded from the data analysis.1Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County,IL; IL2 = Stark County, IL,IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS;MI = Ottawa County, MI; MO2 =Lincoln County, MO,MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA; WI =Walworth County,WI.2Flower color and plant pubescence data were categorical and were notstatistically analyzed.S.E. = standard error. Means based on n = 3.Monsanto Company 09-SY-201UPage 350 of 471Table G-3 (cont.). Phenotypic Comparison of MON 87705to the Conventional Soybean Control within Each SitePhenotypic Characteristic (units)Plant height(in)Lodging(0-9 scale)Pod Shattering(0-9 scale) Final stand count (#/plot)Seed moisture (%)Site1MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)AR 25.0 (0.6) 24.1 (0.5)2.3 (0.3) 1.0 (0.6)0.7 (0.3) 0.3 (0.3)98.0* (12.1) 153.7 (16.5) 10.5 (0.6) 9.6 (0.1)IA1 40.3* (0.1) 42.9 (0.6)5.0 (0.6) 5.0 (0.6)0.0 (0.0) 0.3 (0.3)172.8* (12.5) 213.0 (8.2) 10.6 (0.1) 10.7 (0.0)IA2 42.7 (1.1) 44.3 (1.7)4.3 (0.3) 4.3 (0.3)0.7 (0.3) 0.7 (0.3)238.3 (11.3) 257.7 (8.4)12.4 (0.4) 12.3 (0.1)IL1 24.7 (0.3) 25.1 (0.4)0.0‡ (0.0) 0.0 (0.0)0.0‡ (0.0) 0.0 (0.0)254.3* (1.8) 276.0 (2.0)9.3 (0.1) 8.9 (0.1)IL2 41.3 (0.9) 43.1 (0.5)3.7 (0.7) 3.7 (0.3)0.0‡ (0.0) 0.0 (0.0)206.6* (8.9) 248.1 (9.7) 13.2 (0.2) 12.8 (0.6)IL3 42.8 (2.3) 43.5 (0.6)2.3 (0.3) 2.3 (0.3)0.0‡ (0.0) 0.0 (0.0)264.7 (18.0) 293.3 (12.7) 9.7 (0.4) 9.5 (0.6)IN1 33.0 (2.0) 33.3 (0.6)0.0‡ (0.0) 0.0 (0.0)0.3 (0.3) 0.0 (0.0)226.3* (19.3) 255.7 (11.2) 13.8 (0.2) 13.9 (0.1)IN2 35.4* (1.0) 33.6 (0.6)4.0* (0.6) 1.7 (0.3)0.0‡ (0.0) 0.0 (0.0)231.7 (19.6) 236.0 (6.4) 13.9 (0.5) 13.9 (0.5)KS 31.2 (0.6) 31.1 (0.1)0.3 (0.3) 1.0 (0.0)0.0 (0.0) 0.0 (0.0)139.3 (16.6) 157.3 (7.3) 11.8* (0.3) 13.4 (1.0)MI 30.5 (1.3) 31.3 (2.1)0.0 (0.0) 0.0 (0.0)0.0‡ (0.0) 0.0 (0.0)285.0 (3.8) 290.3 (2.9) 13.6 (0.4) 13.0 (0.1)MO2 32.4 (0.4) 32.4 (0.9)0.0‡ (0.0) 0.0 (0.0)0.0‡ (0.0) 0.0 (0.0)288.6 (7.3) 302.4 (6.6) 15.0 (0.2) 13.7 (0.4)MO3 29.6* (0.5) 32.0 (0.4)0.0‡ (0.0) 0.0 (0.0)0.0‡ (0.0) 0.0 (0.0)241.0 (17.8) 285.0 (3.6) 12.0* (0.8) 14.4 (0.2)MO4 29.4 (0.8) 30.2 (1.2)0.3 (0.3) 0.7 (0.3)0.3 (0.3) 0.3 (0.3)189.7 (12.1) 202.7 (7.2) 10.6 (0.2) 10.5 (0.0)NE 39.3 (0.4) 39.4 (1.4)0.0‡ (0.0) 0.0 (0.0)0.0‡ (0.0) 0.0 (0.0)300.0 (2.3) 286.7 (5.8) 14.1 (0.2) 14.2 (0.0)OH 30.0 (0.6) 33.3 (1.8)0.0 (0.0) 0.7 (0.3)0.0‡ (0.0) 0.0 (0.0)116.7* (7.7) 156.7 (3.4) 12.0 (0.3) 11.9 (0.2)PA 28.6* (0.0) 32.0 (0.6)0.3 (0.3) 0.3 (0.3)0.0‡ (0.0) 0.0 (0.0)253.7 (7.8) 268.7 (3.5) 15.1 (0.5) 14.7 (0.6)WI 45.3 (0.1) 45.0 (1.4)7.0 (0.0) 6.3 (0.7)1.0‡ (0.0) 1.0 (0.0) 170.3 (11.6) 185.7 (10.5) 11.6 (0.0) 11.4 (0.1)* Indicates a statistically significantdifference between MON 87705 and theconventional soybeancontrol (p < 0.05).‡ Indicates a lack of variability in the data which precluded statistical analysis.1Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County,IL; IL2 = Stark County, IL,IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS;MI = Ottawa County, MI; MO2 =Lincoln County, MO,MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA; WI =WalworthCounty, WI.- Data not available or excluded from the data analysis.S.E. = standard error. Means based on n = 3.Monsanto Company 09-SY-201UPage 351 of 471Table G-3 (cont.). Phenotypic Comparison of MON 87705to the Conventional Soybean Control within Each SitePhenotypic Characteristic (units)100 seed weight (g)Test weight (lb/bu)Yield (bu/ac)Site1MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)MON 87705(S.E.)Control(S.E.)AR 15.1 (0.1) 16.0 (0.2)53.4 (0.1) 53.3 (0.7)40.8 (8.9) 36.1 (7.5)IA1 23.8 (0.0) 24.3 (0.2) 53.1 (1.7) 50.9 (1.2) 69.0 (3.8) 72.0 (2.0)IA2 13.8* (0.1) 14.4 (0.2) 55.2 (0.2) 55.0 (0.0) 75.6 (8.5) 68.1 (1.0)IL1 13.7 (0.9) 15.2 (0.3) 57.0 (1.0) 56.0 (0.6) 24.4 (4.4) 24.5 (2.2)IL2 13.5 (0.1) 13.7 (0.6)53.3 (0.9) 52.3 (0.3)43.5* (0.5) 51.2 (2.3)IL3 14.1 (0.3) 14.0 (0.4) 53.7 (0.8) 55.6 (0.9) 77.1 (3.5) 78.8 (5.4)IN1 15.3 (0.3) 15.9 (0.2) 50.1* (1.6) 42.1 (0.6) 56.2 (1.7) 66.0 (5.0)IN2 16.5 (0.1) 16.7 (0.5) 46.4 (0.6) 46.3 (0.1) 71.9 (0.9) 68.1 (3.9)KS 13.8 (0.1) 14.1 (0.2) 56.3 (0.7) 56.0 (0.7) 65.9 (6.1) 63.6 (2.8)MI 19.6* (0.2) 21.3 (0.6) 56.7 (0.3) 57.6 (0.2) 54.6 (1.3) 52.1 (1.3)MO2 14.8 (0.1) 14.9 (0.2) 59.7 (0.3) 60.0 (0.5) 31.4 (1.5) 28.9 (6.9)MO3 14.9 (0.5) 13.9 (0.6) 59.7 (0.5) 58.7 (0.2) 21.7* (0.8) 31.8 (1.1)MO4 13.3* (0.3) 14.5 (0.4) 50.7* (1.2) 53.8 (0.4) 63.2 (4.0) 58.2 (1.9)NE 15.0 (0.1) 14.9 (0.1) 59.0 (0.1) 59.0 (0.0) 66.1 (2.8) 67.8 (3.7)OH 16.0 (0.4) 16.9 (0.1) 47.4 (0.6) 48.7 (1.0) 52.0 (4.3) 50.2 (1.6)PA 16.3 (0.9) 16.7 (0.3) 55.8 (0.6) 55.8 (0.8) 54.6 (2.9) 61.0 (1.5)WI N/A N/A56.0‡ (0.0) 56.0 (0.0)55.6 (1.9) 52.8 (1.0)* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05).‡ Indicates a lack of variability in the data which precluded statistical analysis.N/A = Data not available or excluded from the data analysis.1Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County,IL; IL2 = Stark County, IL,IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS;MI = Ottawa County, MI; MO2 =Lincoln County, MO,MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA; WI =WalworthCounty, WI.S.E. = standard error. Means based on n = 3.Monsanto Company 09-SY-201UPage 352 of 471Table G-4. Growth Stage Monitoring of MON 87705, the Conventional Soybean Control, and the Reference SoybeanVarietiesDate and Range of Growth Stages Observed2Site1SubstanceObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs.5 Obs. 6 Obs. 7 Obs. 8 Obs. 9AR 06/20/2007 07/09/2007 07/27/2007 08/13/2007 09/04/2007 09/25/2007———MON 87705 V2-V3 V8-V9 R4-R5 R5-R6 R7 R8———Control V3 V8-V9 R4-R5 R6 R7 R8———References V2-V3 V7-V9 R4-R5 R5-R6 R7 R8———IA1 06/18/2007 07/09/2007 07/30/2007 08/20/2007 09/11/2007 10/02/2007———MON 87705 V3 V8 R3 R5 R6-R7 R8———Control V3 V8 R3 R5 R6-R7 R8———References V3 V8 R3 R5 R6-R7 R8———IA2 06/20/2007 07/10/2007 07/31/2007 08/17/2007 09/05/2007 09/23/2007 10/11/2007——MON 87705 V2 V6 R3-R4 R5 R6 R7 R8——Control V2 V7 R3 R5 R6 R7 R8——References V2 V6-V7 R3-R4 R5 R6 R7 R8——IL1 06/20/2007 07/19/2007 08/06/2007 08/27/2007 09/10/2007 09/27/2007———MON 87705 V2 R1 R3 R6 R6 R8———Control V2-V3 R1 R3 R6 R6-R7 R8———References V2-V3 R1 R3 R6 R6 R7-R8———IL2 06/27/2007 07/11/2007 07/24/2007 08/08/2007 08/27/2007 09/17/2007 10/08/2007——MON 87705 V2 V3-V4 R1-R2 R2-R3 R5 R7 R7-R8——Control V2 V3-V4 R1-R2 R3-R4 R5 R7 R7-R8——References V2 V3-V4 R1-R2 R2-R4 R5 R7 R7-R8——1Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County,IL2 = Stark County, IL.2Obs. = Observation number; dates in month/day/year format.- Indicates information not available.Monsanto Company 09-SY-201UPage 353 of 471Table G-4 (continued). Growth StageMonitoring of MON 87705, the Conventional Soybean Control, and the ReferenceSoybean VarietiesDate and Range of Growth Stages Observed2Site1SubstanceObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs.5 Obs. 6 Obs. 7 Obs. 8 Obs. 9IL3 06/05/2007 06/20/2007 07/11/2007 07/27/2007 08/10/2007 08/30/2007 09/18/2007 10/04/2007—MON 87705 VE V3 R1-R2 R3 R5 R6 R7 R8—Control VE V3 R2 R3 R5 R6 R7 R8—References VE V3 R1-R2 R3 R5 R6 R7 R8—IN1 06/26/2007 07/16/2007 07/25/2007 08/17/2007 09/12/2007 10/17/2007———MON 87705 V2-V3 R1 R1-R2 R3-R4 R6 R8———Control V2-V3 R1 R1-R2 R3-R4 R6 R8———References V2-V3 R1 R1-R2 R3-R4 R6-R7 R8———IN2 07/02/2007 07/27/2007 08/01/2007 08/16/2007 09/12/2007 09/28/2007 10/19/2007——MON 87705 V2 R1 R2-R4 R5 R6 R8 R8——Control V2 R1 R2-R4 R5 R6 R8 R8——References V2 R1 R2-R3 R5 R6 R8 R8——KS 06/12/2007 06/29/2007 07/13/2007 08/01/2007 08/13/2007 08/24/2007 09/05/2007 10/05/2007—MON 87705 V3 V8 R2 R5 R5 R6 R7 R8—Control V3 V8 R2 R5 R5 R6 R7 R8—References V3 V8 R2 R5 R5 R6 R7 R8—MI 06/20/2007 07/05/2007 07/18/2007 08/01/2007 08/14/2007 08/29/2007 09/12/2007 09/26/2007 10/09/2007MON 87705 V2 V5 R1-R2 R3 R5 R5 R6 R7 R8Control V2-V3 V5 R2 R3 R5 R5 R6 R7 R8References V2-V3 V5-V6 R1-R2 R2-R3 R5 R5 R6 R7 R81Site codes are as follows: IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; MI= Ottawa County, MI.2Obs. = Observation number; dates in month/day/year format.- Indicates information not available.Monsanto Company 09-SY-201UPage 354 of 471Table G-4 (continued). Growth StageMonitoring of MON 87705, the Conventional Soybean Control, and the ReferenceSoybean VarietiesDate and Range of Growth Stages Observed2Site1Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9MO2 06/26/2007 07/13/2007 07/25/2007 08/05/207 08/26/2007 09/17/2007 — — —MON 87705 V2-V3 V6-V8 R3-R4 R5-R6 R6 R8 — — —Control V2-V3 V7-V8 R3-R4 R5 R6 R8 — — —References V2-V3 V7-V9 R3-R4 R5-R6 R6 R7-R8 — — —MO3 06/06/2007 06/21/2007 07/09/2007 07/24/2007 08/05/2007 08/26/2007 09/17/2007 — —MON 87705 VC V2 V7-V8 R4 R5 R6 R8 — —Control VC V2 V7-V8 R4 R5 R6 R8 — —References VC V2 V7-V8 R4 R5-R6 R6-R7 R8 — —MO4 06/29/2007 07/13/2007 08/01/2007 08/14/2007 09/11/2007 10/08/2007 — — —MON 87705 V2-V3 V6-V7 R3 R5 R6 R8 — — —Control V3 V6-V7 R3 R5 R6 R8 — — —References V2-V3 V6-V7 R2-R3 R5 R6 R8 — — —NE 06/13/2007 07/02/2007 07/23/2007 08/13/2007 09/05/2007 09/25/2007 — — —MON 87705 V2 V6 R3 R5 R6 R7 — — —Control V2 V6 R3 R5 R6 R7 — — —References V2 V6R3 R5 R6 R7 — — —OH 06/15/2007 07/02/2007 07/20/2007 08/14/2007 09/04/2007 09/25/2007 — — —MON 87705 V1-V2 V3-V4 R1-R2 R3-R4 R6 R8 — — —Control V1-V2 V3-V4 R1-R2 R3-R4 R6 R8 — — —References V1-V2 V3-V4 R1-R2 R3-R4 R6 R8 — — —1Site codes are as follows: MO2 = Lincoln County, MO, MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE;OH = FayetteCounty, OH.2Obs. = Observation number; dates in month/day/year format- Indicates information not available.Monsanto Company 09-SY-201UPage 355 of 471Table G-4 (continued). Growth StageMonitoring of MON 87705, the Conventional Soybean Control, and the ReferenceSoybean VarietiesDate and Range of Growth Stages Observed2Site1Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9PA 06/29/2007 07/13/2007 07/27/2007 08/13/2007 08/31/2007 09/19/2007 10/10/2007 — —MON 87705 V1 V4 V7 R4 R5-R6 R6 R8 — —Control V1 V4 V7-V8 R4 R5-R6 R6 R8 — —References V1 V4 V7-V8 R3-R4 R5-R6 R6 R8 — —WI 06/20/2007 07/11/2007 08/16/2007 08/31/2007 09/27/2007 10/24/2007 — — —MON 87705 V2 V6 R3-R4 R5 R6 R8 — — —Control V2 V6 R3-R4 R5 R6 R8 — — —References V2 V6-V7R3-R4 R5 R6 R8 — — —1Site codes are as follows:PA = Berks County, PA; WI = Walworth County, WI.2Obs. = Observation number; dates in month/day/year format.- Indicates information not available.Monsanto Company 09-SY-201U Page 356 of 471 Table G-5. Abiotic Stressor Evaluation Us ing Observational Se verity Scale for
MON 87705 and the Conventional Soybean Control

Abiotic stressor Number of observations

across the sites Number of observations

where no differences were

detected between MON 87705

and the control

Total 167 167

Chloride toxicity 4 4

Cold 2 2
Drought 37 37

Flood 3 3

Frost 4 4

Hail 30 30

Heat stress 27 27

Moisture stress 4 4
Nutrient deficiency 12 12
Soil compaction 8 8

Wet soil 4 4

Wind 32 32

Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2, Observation 3 = R3-R5, and Observation 4 = R6-R8. No differences were observed between MON 87705 and the control during any observation. Data were not subjected to statistical analysis.Monsanto Company 09-SY-201U Page 357 of 471 Table G-6. Disease Damage Evaluations Usin g an Observational Severity Scale for
MON 87705 and the Conventional Soybean Control

Disease Number of observations

across the sites Number of observations

where no differences

were detected between

MON 87705 and the control

Total 206 203

Alternaria leaf spot 14 14

Anthracnose 2 2

Asian rust 3 3
Bacterial blight 22 19*

Brown stem rot 5 5

Brown stem rust 1 1

Charcoal rot 2 2
Downy mildew 14 14
Cercospora 6 6
Frogeye leaf spot 31 31
Phytophthora 1 10 10
Powdery mildew 11 11
Pythium 7 7

Rhizoctonia 2 8 8

Sclerotinia 2 2
Seedling blight 1 1
Septoria (brown spot) 25 25
Soybean cyst nematode 1 1

Sudden death 12 12
Soybean mosaic virus 9 9

White mold 9 9
Soybean rust 11 11

*Indicates a difference observed between test and the control for bacterial blight at MO2 site (slight vs. none; Observation 1, Observation 2, and Observation 3). Data were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stag es: Observation 1 = V2-V4, Observation 2 = R1-R2, Observation 3 = R3-R5, and Observation 4 = R6-R8.

1 includes Phytophthora root rot.

2 includes Rhizoctonia root rot.Monsanto Company 09-SY-201U Page 358 of 471 Table G-7. Arthropod Damage Evaluated Usin g an Observational Severity Scale for
MON 87705 and the Conventional Soybean Control

Arthropod Number of

observations across

the sites Number of observations

where no differences were

detected between MON 87705

and the control

Total 206 204
Aphids 1 28 27* Armyworms 7 7

Bean leaf beetles 46 46

Flea beetles 1 1 Grasshoppers 26 26

Green cloverworms 20 20 Japanese beetles 28 28 Leafhoppers 2 14 13*

Leafrollers 3 3

Mexican bean beetles 3 3 Soybean loopers 5 5 Spider mites 4 4 Stink bugs 11 11 Thistle caterpillars 2 2 Thrips 1 1

Whiteflies 2 2

Wireworms 1 1 Yellow wooly bear 4 4

*Indicates a difference observed between MON 87705 and the control for aphids (none vs. slight; Observation 2) and leafhoppers (none vs . slight; Observation 2) at the WI site. Data were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2, Observation 3 = R3-R5, and Observation 4 = R6-R8. Data were not subjected to statistical analysis.

1 Includes soybean aphids.

2 Includes potato leafhoppers.Monsanto Company 09-SY-201UPage 359 of 471Table G-8. Abundance of Pest Arthropodsin Beat Sheet Samples Collected from MON 87705, the Conventional SoybeanControl, and the Reference Soybean VarietiesAbundance of Pest Arthropods2Collection 1 Collection 2 Collection 3Arthropod Site1MON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeAphid IL2 31.7 (31.7) 18.3 (14.5) 5.0 – 25.3 — — — — — —MO2 — — — 145.0 (123.2) 35.3 (32.4) 16.7 – 199.0 — — —Bean leafbeetleIA1 0.0 (0.0) 0.3 (0.3) 0.0 – 1.0 1.3 (0.3) 0.7 (0.3) 0.0 – 1.3 0.0* (0.0) 1.7 (1.2) 0.0 – 2.0IL2 12.0 (3.5) 12.0 (2.6) 8.7 – 12.0 11.0 (1.0) 10.3 (2.9) 7.3 – 16.0 17.0 (0.6) 22.7 (10.9) 8.7 – 21.3IN1 3.7 (1.5) 2.0 (1.0) 1.7 – 3.0 1.0 (0.6) 0.0 (0.0) 0.3 – 1.0 62.0 (18.0) 71.3 (17.9) 37.0 – 135.3MO2 3.7(1.2) 2.3(0.7) 2.3 – 5.0 0.7(0.3) 0.7(0.7) 0.0 – 1.3 4.7 (2.2) 3.3 (1.7) 2.0 – 5.0Corn fleabeetleIN1 — — — — — — 0.3 (0.3) 0.0 (0.0) 0.3 – 1.7GreencloverwormIA1 1.0 (0.0) 1.7 (0.3) 0.7 – 1.3 1.7 (1.2) 0.3 (0.3) 0.0 – 1.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.0IL2 0.0†(0.0) 0.0 (0.0) 0.0 – 0.0 2.0 (0.6) 1.3 (0.9) 0.0 – 2.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3IN1 0.7 (0.3) 0.0 (0.0) 0.0 – 0.3 5.7 (2.0) 8.3 (2.6) 1.7 – 7.3 0.0 (0.0) 1.3 (1.3) 0.0 – 1.0MO2 7.7 (1.8) 4.3 (1.7) 6.7 – 8.7 3.3 (2.4) 1.7 (1.7) 1.0 – 5.3 1.3 (0.9) 2.7 (1.8) 0.3 – 1.7JapanesebeetleIN1 0.3 (0.3) 1.3 (0.7) 0.0 – 2.0 2.3 (1.9) 2.0 (0.6) 0.7 – 4.7 — — —PotatoleafhopperIA1 1.3 (0.3) 1.0 (1.0) 0.0 – 2.7 — — — — — —IL2 16.0 (3.5) 30.0 (3.5) 25.7 – 117.3 — — — — — —IN1 2.3 (1.5) 5.7 (2.8) 0.0 – 5.7 2.3 (2.3) 2.3 (0.3) 3.0 – 8.3 — — —* Indicates a statistically significant difference between MON 87705 and the control (p < 0.05).Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections performed at three cropdevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8.1Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO.2MON 87705 and control values represent mean number ofarthropods collected across three replications. S.E. = standard error. Means based on n = 3.†No statistical comparisons were made due to lack of variability in the data.- Indicates the arthropod was not evaluated.Monsanto Company 09-SY-201UPage 360 of 471Table G-8 (continued). Abundance of PestArthropods in Beat Sheet Samples Collected from MON 87705, the ConventionalSoybean Control, and the Reference Soybean VarietiesAbundance of Pest Arthropods2Collection 1 Collection 2 Collection 3Arthropod Site1MON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeStink bug IA1 0.3(0.3) 0.0 (0.0) 0.0 – 1.0 0.0 (0.0) 0.7 (0.7) 0.0 – 1.0 0.3 (0.3) 0.0 (0.0) 0.0 – 1.3IL2 0.3 (0.3) 0.0 (0.0) 0.0 – 0.7 2.0 (1.0) 0.7 (0.7) 1.3 – 3.7 2.7 (0.9) 1.3 (0.9) 2.0 – 3.3IN1 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 1.0 (0.0) 0.3 (0.3) 0.0 – 0.3 4.3 (1.9) 6.0 (1.2) 1.0 – 3.7MO2 0.3 (0.3) 0.3 (0.3) 0.0 – 1.0 0.3 (0.3) 1.0 (0.6) 0.0 – 1.7 1.0 (1.0) 3.0 (1.0) 1.0 – 3.0Thrips IA1 23.0 (3.1) 11.7 (6.6) 5.0 – 29.3 — — — — — —MO2 42.0 (21.0) 66.0 (54.9) 15.3 –34.0 — — — — — —Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections performed at three cropdevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8.1Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO.2MON 87705 and control values represent mean number ofarthropods collected across three replications. S.E. = standard error. Means based on n = 3.- Indicates the arthropod was not evaluated.Monsanto Company 09-SY-201UPage 361 of 471Table G-9. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87705, the ConventionalSoybean Control, and the Reference Soybean VarietiesAbundance of Beneficial Arthropods2Collection 1 Collection 2 Collection 3ArthropodSite1MON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeBig-eyed bug MO2 1.0 (0.6) 1.0 (0.6) 0.3 – 3.0 5.3 (2.3) 7.7 (3.2) 8.0 – 9.0 7.3 (6.4) 8.0 (2.5) 10.7 – 19.7CarabidbeetleIL2 — — — — — — 0.3 (0.3) 0.3 (0.3) 0.3 – 1.0IN1 — — — — — — 0.7 (0.7) 0.3 (0.3) 0.0 – 9.3Lacewing IA1 — — — 0.3(0.3) 0.3 (0.3)0.0 – 0.7 — — —MO2 — — — 1.0 (0.6) 1.0 (1.0) 0.7 – 1.7 0.0 (0.0) 0.3 (0.3) 0.0 – 1.3LadybirdbeetleIA1 — — — 0.7 (0.3) 0.3 (0.3) 0.0 – 1.0 — — —IL2 — — — 0.0 (0.0) 0.7 (0.7) 0.3 – 4.3 0.7 (0.7) 0.7 (0.7) 0.0 – 2.0MO2 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 1.7 (1.7) 0.7 (0.7) 0.3 – 3.0 — — —MicroparasitichymenopteraIL2 — — — 1.3 (0.7) 0.7 (0.3) 0.7 – 2.3 — — —NabisIA1 0.3 (0.3) 2.3 (1.9) 0.7 – 2.0 2.0 (1.2) 2.0 (0.6) 1.0 – 2.7 0.3 (0.3) 0.3 (0.3) 0.0 – 0.3IL2 0.0 (0.0) 0.7 (0.3) 0.0 – 2.3 0.7 (0.3) 1.3 (0.7) 0.3 – 2.3 0.7 (0.7) 1.0 (1.0) 0.0 – 1.0IN1 0.3 (0.3) 0.3 (0.3) 0.0 – 1.3 1.0 (0.0) 1.3 (0.7) 0.3 – 2.0 5.0 (2.0) 3.7 (1.9) 2.3 – 16.0MO2 0.3 (0.3) 1.0 (0.6) 0.3 – 2.3 1.3 (0.9) 1.7 (0.3) 0.0 – 1.7 0.3 (0.3) 0.3 (0.3) 0.3 – 1.3OriusIA1 0.3 (0.3) 0.0 (0.0) 0.0 – 0.3 0.3 (0.3) 0.3 (0.3) 0.0 – 0.7 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3IL2 25.7 (15.7) 23.3 (4.8) 21.3 – 35.0 32.0 (4.0) 32.0 (7.5) 25.3 – 65.0 4.0 (1.0) 4.7 (2.0) 5.0 – 12.0IN1 3.0 (0.6) 5.7 (1.7) 1.0 – 3.3 14.3 (5.2) 17.3 (0.9) 8.7 – 23.0 7.3 (3.3) 1.3 (0.9) 1.0 – 31.3MO2 1.0 (1.0) 2.0 (2.0) 0.0 – 1.3 3.3 (1.8) 1.3 (0.7) 1.0 – 4.0 0.0 (0.0) 0.3 (0.3) 0.0 – 0.3Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections performed at three cropdevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8.1Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO.2MON 87705 and control values represent mean number ofarthropods collected across three replications. S.E. = standard error. Means based on n = 3.- Indicates arthropod not evaluated.Monsanto Company 09-SY-201UPage 362 of 471Table G-9 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87705, theConventional Soybean Control, and the Reference Soybean VarietiesAbundance of Beneficial Arthropods2Collection 1 Collection 2 Collection 3Arthropod Sites1MON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87705Mean (S.E.)ControlMean (S.E.)ReferenceRangeSpider IA1 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 0.3 (0.3) 1.0 (1.0) 0.0 – 0.3 0.0 (0.0) 0.0 (0.0) 0.0 – 1.0IL2 1.7 (0.9) 1.3 (0.7) 0.7 – 3.3 1.0 (1.0) 1.7 (0.3) 1.0 – 2.3 0.0 (0.0) 1.7 (0.9) 1.3 – 2.7IN1 0.3 (0.3) 1.0 (0.6) 0.0 – 2.3 0.3 (0.3) 0.3 (0.3) 0.3 – 1.0 1.7 (0.9) 2.7 (0.3) 0.3 – 3.7MO2 0.3 (0.3) 0.7 (0.3) 0.0 – 2.0 1.3 (0.3) 2.0 (0.6) 0.0 – 2.3 2.0 (1.2) 3.3 (0.7) 0.3 – 3.7Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections performed at three cropdevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8.1Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO.2MON 87705 and control values represent mean number ofarthropods collected across three replications. S.E. = standard error. Means based on n = 3.- Indicates arthropod not evaluated.Monsanto Company 09-SY-201U Page 363 of 471 Appendix H. Materials and Methods for Pollen Morphology and Viability

Evaluation

H.1 Plant Production

MON 87705, a conventional soybean control (A 3525), and five commercially available reference soybean varieties were grown in St. Louis County, MO, in a randomized complete block design with three replicatio ns. Each plot consisted of four rows approximately 20 ft in length.

H.2 Flower Collection

When soybean plants were at flowering stag e, whole flowers were collected from five non-systematically selected plants from the fi rst and fourth row of each plot. All flowers from all plots were collected on the same day. Four flowers were collected from each of the five plants per plot: one flower from the bottom, two flowers from the middle, and one flower from the top of each plant. Up to five additional flower s were collected from each plot to ensure a sufficient quantity of pollen for evaluation. All flowers selected from a plot were transferred into a single, clean container and labell ed with the plot number from which the sample originated, the entry number, and the entry name. The containers were kept on wet ice or refrig erated for less than eight hours until the pollen was prepared and stained.

H.3 Pollen Sample Preparation

Pollen samples were prepared in a laboratory. Clean microscope slides were labelled with the plot number. A circle of approxima tely 1 cm diameter was drawn in the center of the slide with a pap hydr ophobic barrier pen. Tweezers a nd a dissecting needle were used to open each of the collected flowers from a plot and brush the pollen into the circle

on the slide. The tweezers were cleaned be tween extractions. Approximately 20 μl of Alexander’s stain (Alexander, 1980) was added to the center of the circle containing the pollen. The pollen was stained at ambient te mperature for at least ten minutes prior to examination. Pollen samples from all plots within a replicate were stained and evaluated on the same day.

H.4 Data Collection

Pollen characteristics were assessed by vi ewing samples under an Olympus Provis AX70 light/fluorescence microscope equipped with an Olympus DP70 digital color camera. The microscope and camera were connected to a computer runni ng Microsoft Windows 2000 Professional (© 1981-1999, Microsoft Corp.) and installed with associated camera software (DP Controller v1.2.1.108 and DP Manager v1.2.1.107 [Olympus Optical Co., Ltd.] and imaging software Image-Pro Plus v4.5.1.27 [Media Cybernetics, Inc.]).

Pollen Viability: When exposed to the stain so lution, viable pollen grains stained red to purple due to the presence of liv ing cytoplasmic content. Nonvi able pollen grains stained blue to green and may have appeared round to collapse in shape, depending on the degree of hydration. For each pollen sample, the num ber of viable and nonviable pollen grains

was counted from a minimum of 75 pollen grains from a ra ndom field of view under theMonsanto Company 09-SY-201U Page 364 of 471 microscope. Dense clusters of pollen or polle n grains adhering to fl ower parts were not counted because they may not have ab sorbed the stain solution uniformly.

Pollen Diameter: Micrographs (400X resolution) of ten representative pollen grains from each plot were taken and imported into the im aging software. The so ftware was used to measure pollen grain diameter along two pe rpendicular axes for each selected pollen grain. Mean pollen diameter for each plot was calculated from the 20 total

measurements.

General Pollen Morphology: Genera l pollen morphology was observed from micrographs of MON 87705, the conventiona l soybean control, and commercial reference soybean varieties that were also used for pollen diameter measurements.

H.5 Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS (SAS, 2002-2003). The level of sta tistical significance was predetermined to be 5% (p<0.05). MON 87705 was compared to the conventional soybean control for percent viable pollen and pol len diameter. No statisti cal comparisons were made between MON 87705 and the reference soybean va rieties. Instead, a reference range for

each measured characteristic was determ ined from the minimum and maximum mean values from among the reference soybean va rieties. General pollen morphology was qualitative; therefore, no stat istical analysis was conducte d on these observations.

Appendix H References

Alexander, M.P. 1980. A vers atile stain for pollen fungi, yeast and bacteria. Stain Technology 55:13-18.

SAS. 2002-2003. SAS Software Re lease 9.1 (TS1M0), SAS Inst itute, Inc., Cary, North

Carolina.Monsanto Company 09-SY-201U Page 365 of 471 Appendix I. Materials and Methods for Symbiont Evaluation

I.1 Materials

The starting seed of MON 87705 was produc ed in Puerto Rico in 2006-2007 under Protocol LS272 by Monsanto Tr ait Development. The control substance starting seed was produced in Illinois in 2007 under Monsanto Production Plan 07-01-83-18. Reference substance starting seed was obt ained from commerci al sources or was produced in Puerto Rico in 2007 under Seed Increas e 07-SI-71-01. Nodu les, root tissue, and shoot tissue collected from MON 87705, the conventional soybean control, and reference soybean varieties were evaluated in the study.

Table I-1 Starting seed of MON 87705, Control, and Commercial Soybean
Reference Varieties Used in the Symbiont Evaluation

Materials Material Type Material Source 1 Phenotype

MON 87705 Test Monsanto TD Improved fatty acid profile

A3525 Control 07-01-83-18 Conventional

A2553 Reference Commercial Conventional

Stine 3300-0 Reference 07-SI-71-01 Conventional

Stewart SB3454 Reference Commercial Conventional

Garst 3585N Reference Commercial Conventional

Hartz H5218 Reference 07-SI-71-01 Conventional

A5560 Reference 07-SI-71-01 Conventional

1 Starting seed were obtained from commercial so urces, Monsanto Trait Development (TD), Monsanto Field Production 07-01-83-18, or Monsanto Seed Increase 07-SI-71-01.

The presence or absence of MON 87705 in the te st and control starting seed was verified by event-specific polymerase chain reaction (P CR) analyses. Results of PCR analyses were as expected.

I.2 Greenhouse Phase and Experimental Design

MON 87705, the conventional soybe an control, and reference soybean varieties starting seed were planted in 6-inch pots containi ng nitrogen-deficient potting medium (Sunshine Mix #2 Basic/LB2, Sun Gro Horticulture, Inc., Garland, TX) comprised of primarily peat, vermiculite, and perlite. Plants from MON 87705, the conventional soybean control, and reference soybean varieties starting seed were grown in a greenhouse where actual temperatures ranged from approximately 21 o C to approximately 42 o C. Eight replicate pots were planted with three seeds per pot for each of MON 87705, the conventional soybean control, and reference soybean varietie s. At planting, each seed was inoculatedMonsanto Company 09-SY-201U Page 366 of 471 with approximately 1 x 10 7 cells of Bradyrhizobium japonicum (VAULT NP, Becker Underwood, Ames, IA) in phosphate-buffered salin e. Pots were arra nged after thinning in eight replicated blocks for the 6-week sampling period using a randomized complete block design.

The reference soybean varieties starting seed were planted on September 9 and 10, 2008,

and MON 87705 and the conventio nal soybean control startin g seed were planted on September 11, 2008. In all cases, replicat e pots had a minimum of one plant emerge within one week. A solution of nitrogen -free nutrient solution (~250 ml) was added weekly after plant emergence.

I.3 Plant Harvesting/Data Collection

Six weeks after emergence, plants were exci sed at the surface of the potting medium and shoot and root plus nodule mate rial were removed from the pot s. The shoot material was cut into smaller pieces and pl aced in labelled bags. The plant roots with nodules were separated from the potting medium by washi ng with water. Excess moisture was removed using absorbent paper towels and the roots plus nodules were placed in labeled bags. The same day that plants were harv ested, nodules were removed by hand from the roots of each plant, enumerated, and the fr esh weight (fwt) was determined. Nodules from each plant were then dried for at least 72 hours at approximately 65 °C, and dry weights were determined.

The remaining root and shoot mass (fresh wei ght) were determined for each plant. Root and shoot material from each plant was then dried for at least 72 hours at approximately 65 °C for dry weight determination. Th e shoot tissue was ground after drying with a Harbil 5G high-speed paint shak er prior to total nitrogen an alysis. Shoot total nitrogen was determined by combustion using a nitrog en analyzer (Rapid N Cube, Elementar Americas, Inc.).

I.4 Statistical Analysis

The data consisted of six m easurement endpoints taken at the six week sampling period: nodule number, nodule dwt (g), sh oot dwt (g), root dwt (g), and shoot total nitrogen (% and g/plant). Data obtaine d from MON 87705, the contro l (A3525), and the reference soybean varieties were analyzed.

An analysis of variance was conducted usi ng a randomized complete block design with eight replications for each test, control, a nd reference substance. Data were analyzed using the Statistical An alysis System (SAS Version 9.2, SAS Institute, Inc. 2002-2008) with the level of statistical significance predetermined to be 5% (p<0.05).Monsanto Company 09-SY-201U Page 367 of 471 Appendix J. Materials and Methods for Analysis of FAD2-1A and FATB1-A RNA

levels in MON 87705 immature seeds

J.1 Test Substance

The test substance was MON 87705. As listed below in the table, four replicates of immature seed from a production plan, ge nerated from seed lot GLP-0702-18254-S were used in this study.

Sample ID Seed Lot # Tissue Type Replicate #

REG07189-0015 GLP- 0702-18254-S Immature seed 1

REG07189-0016 GLP- 0702-18254-S Immature seed 2

REG07189-0017 GLP- 0702-18254-S Immature seed 3

REG07189-0018 GLP- 0702-18254-S Immature seed 4

J.2 Control Substance

The control substance was conventional so ybean variety A3525, the same genetic background as the test substance. As listed below in the table, four replicates of immature seed from a production plan, generated from seed lot GLP-0702-18252-S were used in this study.

Sample ID Seed Lot # Tissue Type Replicate #

REG07189-0001 GLP- 0702-18252-S Immature seed 1

REG07189-0002 GLP- 0702-18252-S Immature seed 2

REG07189-0003 GLP- 0702-18252-S Immature seed 3

REG07189-0004 GLP- 0702-18252-S Immature seed 4

J.3 Reference Substance

Probe templates were used as positive hybridization c ontrols in northern bl ot analyses. Prior to probe generation, DNA segments from the FAD2-1A (Monsanto Sequence Database) and

FATB1-A (Monsanto Sequence Database) genes were generated from conventional soybean genomic DNA using a forward primer specific to the coding region and a reverse primer specific to the 3'UTR region of each gene. The resulting PCR segments of the FAD2-1A and

FATB1-A genes were cloned into pGEM-T Easy vectors (pGEM-T Easy Vector System, Invitrogen, Carlsbad, CA) according to the ma nufacture's manual with minor modifications

documented in the raw data. The modificatio ns were approved by the study director and documented in the raw data. The sequence of the plasmid DNA was confirmed by sequencing and comparison to the expected se quences using pair wise alignments. The plasmid DNAs then were used as templates for PCR reactions to amplify the FAD2-1A and FATB1-A probe templates. The actin probe template was generated by PCR from conventional soybean genomic DNA using a forwar d primer specific to the coding region and a reverse primer specific to the 3'UTR region of the soybean actin gene (Soy57, GI: 1498333). All PCR reactions and purification of DNA segments from agarose gels were performed according to in house SOPs. RiboR ulerTM RNA ladders (high range) fromMonsanto Company 09-SY-201U Page 368 of 471 Fermentas Co (Glen Burnie, MD ) were used for size estimations on northern blots and formaldehyde/agarose gels. The unique identities of the probes, as well as molecular weight

markers are documente d in the raw data.

J.4 Characterization of Test, Control and Reference Substances

The identity of the test and c ontrol substances was determined by event-specific PCR prior to planting the seed in the green house. The st udy director reviewed the chain of custody (COC) documentation and certificate of analysis (COA) to confir m the identity of the test and control substances prior to use of the mate rials in the study and a copy of the COAs was archived with the study data. Immature seed was harvested from th e plants and identity confirmation was performed on each sample by even t-specific PCR prior to use in the study. The study director reviewed the data prior to use of the materials in the study and the raw data was archived with the study. Mature seed was harvested from the plants and tested by an event-specific PCR. These results were reviewed by the study director prior to the start of the study and a copy of the Verification of Identity (VOI) was archived with the study.

Prior to the start of the study, the FAD2-1A, FATB1-A, and actin probe templates were sequenced by the Monsanto Genomics Sequenci ng Center and the sequences were aligned with the expected sequences to confirm the identities of the probes. The raw data are archived with the study.

Extracted RNA and RNA markers were stored in a -80 ° C freezer. DNA probes were stored in

a -20 ° C freezer. The test, control, and reference substances were deemed stable during storage because there were no signs of de gradation on formaldehyde/agarose gels and probing with endogenous genes yielded inte rpretable signals on northern blots.

J.5 Total RNA extraction

Total RNA from test and control substances was extracted according to an in-house SOP.

All extracted RNA was stored in -80 ° C freezers.

J.6 PolyA+ RNA Isolation

PolyA+ RNA 10 from the test and control substances was extracted from total RNA using the Poly(A) Purist MAG Kit (Ambion Co., Austin, TX ) according to the manu facturer's protocol with minor modifications approved by the study director prior to performing the analysis. The procedure was documented in the raw data. The resulting polyA+ RNA was stored in a -80 ° C

freezer.

J.7 Formaldehyde/Agarose Gel Electrophoresis

PolyA+ RNA samples from test and control substances for FAD2-1A northern blot analysis were extracted from approximately 20-50 μg of total RNA, and resolved in 0.8% formaldehyde/agarose gels (0.8% with regard to agarose). PolyA+ RNA samples from test

and control substances for FATB1-A northern blot analyses were extracted from approximately 100 μg total RNA, and resolved on 1.0% form aldehyde/agarose gels (1.0% with regard to agarose).

10 Poly A enriched RNAMonsanto Company 09-SY-201U Page 369 of 471 J.8 Northern Blot Analyses

Northern blot analyses were performed by transferring polyA+ RNA from formaldehyde/ agarose gels onto nylon membranes and prob ing with radiolabeled DNA. FAD2-1A and FATB1-A hybridization signals were stripped from the blots and th e stripped blots were hybridized to a 32 P-labeled actin probe in order to show the relative amount of RNA loaded in each lane. Probes were prepared by random prime labeling. Each probe was added at approximately 1-2 × 10 6 cpm per ml of the hybridization solution.

DNA probe templates were run on the formaldehyde/agarose gels to serve as positive controls for hybridization. On northern blots the probe templates migrated sli ghtly differently than predicted by the RNA ladder. The difference in migration is probably due to the inaccuracy of comparing a DNA probe to an RNA ladder. Th e following table lists the DNA probes, the positive hybridization controls, an d the hybridization and wash te mperatures for each northern blot analysis.

Analysis Positive hybridization

controls Probe Hybridization and Wash Temperature Expression of

FAD2-1A FAD2-1A probe template FAD2-1A 65 oC

Expression of

FATB1-A FATB1-A probe template FATB1-A 60 oC

Expression of

actin actin probe template actin 55 oCMonsanto Company 09-SY-201U Page 370 of 471 Appendix K. Manufacturing Process for Soybean

MON 87705 may be processed using conventi onal industry standard processing methods, which include extrusion methods (Erickson et al., 1980). Following cr acking and aspiration of the soybean to separate the hulls, the hulls are screened to recover the fines generated during cracking, and the cracked soybean meats are conditione d and flaked to rupture oil cells and prep are a thin flake with a large su rface area for solvent extraction. The soybean flakes then undergo solvent extraction with iso- hexane/hexane to yield crude soybean oil and soy meal. The crude oil is processed through a se ries of steps known as refining. In the first step, phospholipids are removed through a pro cess known as degumming. This involves mixing with water to form gums. followed by centrifugation. The fatty acids in the degummed oil are neutralized through the additi on of a caustic solution (sodium hydroxide) to form soaps soluble in water in a process known as alkali-refining . This process is preceded by a pretreatment step with phosphor ic acid. The resul ting soap solution is removed by centrifugation. Water washing an d centrifugation further removes soaps to levels compatible with bleaching. Alternatively, oil can be fu rther treated with trisyl to remove any residual soap content and then filtered. In the next step, the oil is “bleached” by mixing with a citric acid solution, followed by treatment with adsorbent clay to remove the peroxides, phosphatides, color bodies and traces of soap. Under vacuum to inhibit oxidation, the pigments are adsorbed and removed by filtration. In the final refining step, odoriferous components, flavor component s and additional free fatty acids are removed by steam distillation to produce refined, bleached and deodorized (RBD) oil. This process of deodorization is carried out at high temperat ures (250°C) and under vacuum. Anti-oxidants

are utilized to inhibit oxidati on of the oil. A pictorial re presentation of the manufacturing process described above is shown below in Figure K-1.

Figure K-1. Schematic of Manufacturing Process for Soybean Oil. Mill

Ground

Seed Extractor

Seed

Meal Miscella Hexane Stripping Degumming

Caustic Refining

Bleaching

Deodorization Volatiles

RBD Oil Optional PressingMonsanto Company 09-SY-201U Page 371 of 471 Appendix K References

Erickson, D.R., E.H. Pryde, O.L. Brekke, T.L. Mounts and R.A. Falb, eds. 1980. Handbook of Soy Oil Processing and Utili zation. American Soybean A ssociation and American Oil Chemist’s Society, St. Louis, Missouri and Champaign, Illinois. Pp. 49-130.Monsanto Company 09-SY-201U Page 372 of 471 Appendix L. Glyphosate Resistance

L.1 Introduction

Monsanto considers product stew ardship to be a fundamental co mponent of customer service and business practices. Stewards hip of the glyphosate molecule to preserve its usefulness for growers is an important aspect of Monsanto’s stewardship commitments. While herbicide resistance may eventually occur when any he rbicide is widely used, resistance can be postponed, contained and ma naged through research, education and good management practices. These are key elements of Mons anto’s approach to glyphosate stewardship.

The development of plant populations resistant to glyphosate can adversely impact the utility and life cycle of glyphosate prod ucts if it is not managed pr operly. As leaders in the development and stewardship of glyphosate pro ducts for over 30 year s, Monsanto makes a significant investment in research to understa nd and develop the proper use and stewardship of glyphosate. Monsanto also invests in grow er/retailer education a nd training programs to provide information on best practices to mana ge glyphosate resistance. This research

includes an evaluation of factor s that can contribute to the development of weed resistance and develop ways to mitigate the risk. The risk of weeds developing resistance and the potential impact of resistan ce on grower weed management practices vary greatly across different herbicide modes of action and they de pend on a combination of factors. Therefore, there is no single set of best management practices to mitigate resistance for all herbicides, nor is there a single set of best management practices to mitigate resistance to a single herbicide for all weeds in all cropping systems. However, this document provides an overview of Monsanto’s approach to the deve lopment of best management practices to mitigate glyphosate resistance.

L.2 The Herbicide Glyphosate

Glyphosate (N-phosphonome thyl-glycine) (CAS Registry #: 1071-83-6), the active ingredient in the Roundup family of nonselecti ve, foliar-applied, pos temergent agricultural herbicides, is among the world’s most widely us ed herbicidal active ingredients. Glyphosate is highly effective against the majority of economically significant annual and perennial grasses and broadleaf weeds. Currently glyphosate is labeled for control of more than 300 weed species. Glyphosate kills plant cell s by inhibition of 5- enolpyruvylshikimate-3- phosphate synthase (EPSPS), an enzyme involved in the shikimic acid pathway for aromatic amino acid biosynthesis in plants and microorganisms (Franz et al., 1997). Glyphosate is the only known herbicide with this m ode of action (Franz et al., 1997). The relevant aromatic amino acid pathway is not present in mamm alian metabolic system s (Cole, 1985). A comprehensive human safety evaluation and ri sk assessment concluded that glyphosate has low toxicity to mammals, is not a carcinogen, does not adve rsely affect reproduction and development, and does not bioaccumulate in mammals (Williams et al., 2000). An ecotoxicological risk assessm ent concluded that the use of glyphosat e does not pose an unreasonable risk of adverse effects to nontarget species, such as birds and fish, when used according to label directions (Giesy et al., 2000). Glyphosate has favorable environmental characteristics, including a low potential to move through the soil to reach ground water and is degraded over time by soil microbes. Beca use it binds tightly to soil, glyphosate’sMonsanto Company 09-SY-201U Page 373 of 471 bioavailability is reduced im mediately after app lication, which is why glyphosate has no residual soil activity.

L.3 Herbicide Use and H erbicide-resistant Weeds

The vast majority of U.S. soybean growers depe nd, at least in part, on herbicides to control weeds. According to a report by USDA-ERS, herbicides were used on more than 97 percent of the total U.S. soybean acreage in 1997 (Fernandez-Cornejo and McBride, 2002).

Control of weeds in a crop is essential because weeds compet e with the crop for the same limited resources in the field, including sunlight, water and nutrients (Ross and Lembi, 1985; Wilcut et al., 2003). Lack of effective weed control in soybean fields can result in yield

losses of 50-90%. Because failu re to control weeds within th e crop can result in decreased yields and reduced crop quality, an intensive program for weed control is essential to ensure profitability (Da lley et al., 2001). When failure to control weed s in a crop increases weed populations, additional herbicide applications or cultural practices are then required to reduce the weed population.

With any herbicide use, however, comes the poten tial for the selection of weeds resistant to that herbicide. Within a weed species i ndividuals may possess an inherent ability to

withstand the effects of a particular herbicide. Repeated use of that herbicide will expose the weed population to a "sel ection pressure," which may lead to an increa se in the number of surviving resistant individuals in the population (H RAC, 2009). In other words, plants susceptible to the applied herbicide will die, while those few having some type of natural resistance may survive and reproduce.

Weed resistance is generally defined as the natu rally occurring heritable ability of some weed biotypes within a given weed population to survive a herbicid e treatment that should, under normal use conditions, effectively control that weed population. Thus, a resistant weed must demonstrate two criteria as defined by the W eed Science Society of America website at

www.wssa.net : (1) the ability to survive application rates of a herbicide product that once were effective in controlling it, and (2) resist ance is heritable. Procedures to confirm resistance generally require both field and greenhouse analys is. Obtaining a correlation between field and greenhouse studies has been particularly important for the accurate detection of glyphosate resistance, for which the levels of resistance observed have been as low as 2X the susceptible biotypes (Lee a nd Ngim, 2000; Lorraine-Co lwill et al., 1999).

Herbicide-resistant weeds are neither a ne w phenomena nor is resistance unique to glyphosate. In 1952, the first U.S. herbicide- resistant weed, a spreading dayflower species resistant to the synthetic auxin 2,4- D, was identified in Hawaii (Heap, 2009). There are currently 72 herbicide-resistant weed species in the U.S. For example there are 38 weed species resistant to ALS herbicides, 15 species resistant to ACCase inhibitors, 23 weed species resistant to triazine herbicides, and nine (9) weeds species re sistant to glyphosate herbicides (Heap, 2009). Growers have been ma naging these herbicide -resistant weeds for

decades with the use of alternativ e herbicides and/or cultural methods such as tillage or crop

rotation.Monsanto Company 09-SY-201U Page 374 of 471 The occurrence of a herbicide- resistant weed biotype generally does not end the useful lifespan or preclude the effective use of the herb icide in question as part of an overall weed management system. This is particularly tr ue for glyphosate, which remains a very useful tool despite its lack of effectiveness on specifi c resistant weed biotypes due to its ability to control a weed at different growth stages and the wide spectrum of weeds it effectively

controls. Even for other herbicides that do not have the spectrum of control or the wide window of application, growers have continued to have a need to use these products even with the presence of resistant populations . For example, in 1995, imazethapyr and imazaquin, both in the imidazolinone herbicide family, were used on almost 60 percent of the soybean acres in the U.S., even th ough, at that time, ni ne weed species that were resistant to those herbicides were present in soybean producing areas (Heap, 2009).

It is important to distinguish herbicide resistan ce, explained above, from herbicide tolerance. In contrast to species for which resistant biot ypes have occurred, herbicide tolerant weed species has the inherent ability to survive applications of a particular herbicide at recommended rates. In other words, the speci es does not develop tolerance through selection but is innately tolerant to that herbicide. It may also be the case that a certain weed species, while neither resistant nor tolerant, is difficult to control with a particular herbicide, requiring more careful herbicide use and weed manageme nt. Section E herein discusses resistant, tolerant, and hard-to-control species in the context of glyphosate.

L.4 Characteristics of Herbicides a nd Herbicide Use Influencing Resistance

While the incidence of weed resistance is often associated with repeat ed applications of a herbicide product, the actual ons et of resistance w ithin a population depends very much on the specific herbicide chemistry in question, as well as the inherent pr esence of gene(s) that confer the ability of a plant to be resistant to a particular chemical within a specific weed species and even a specific population of that species (Sammons et al., 2007). Some herbicide products are much more prone to develop herbicide re sistance than others (Heap, 2009). Glyphosate has been used extensively for over three d ecades with relatively few cases of resistance development, particularly when compared to many other herbicides, considering the substantial acreage glyphosate-tr eated worldwide, and the total number of weeds that glyphosate can control. The graph in Figure 1 illustrates the instances of weed resistance to various herbicide families. The different slopes observed are largely due to the factors described above, which relate to chemis try and function, in addition to levels of exposure in the field. The summary below descri bes herbicide-specific factors determined to be important in the process of selecting for in dividuals that are inherently resistant to a

herbicide.Monsanto Company 09-SY-201U Page 375 of 471 Figure L-1. Weed Resistance to Vari ous Herbicide Chemical Families

L.5 Mechanisms of Resistance

The application of a herbicide to a weed does not, itself, cause a mutation in later generations of the plant. Rather, over time, those few biot ypes that are not susceptible to the herbicide may become dominant within a population with the repeated use of that herbicide. To date, the three known avenues by which a weed species is resistant to an herbicide have been identified as target site alteration (target site), enhanced metabolism of the herbicides (metabolism), and reduced absorp tion and/or translocation of the herbicide such that the herbicide does not get to the site of action w ithin the plant cell (exclusion) (Sammons et al., 2007).

Herbicide resistance via targ et site is the most common resistance mechanism among the

various herbicide classes. It has been found th at a target site mechanism is the most common mechanism for ALS inhibitors, ACCase inhibito rs, and triazines, bu t is less common for glyphosate. A target site alteration is one where th ere is/are amino acid substitution(s) in the protein that is the target of a herbicide such that the alteration preven ts the binding of the herbicide to the protein and thus the activity of the protein is not alte red and the plant grows normally. For ALS inhibitors, the level of re sistance conferred by a target site mechanism has been found to be as high as 3,400 X (Ferguson et al., 2001). (Note: X is the labeled or recommended rate for a herbicide on a partic ular weed species.) For glyphosate, species found to exhibit a target site m echanism often show low levels of resistance (2-3X) due to the fact that glyphosate is a true transition state inhibitor (Sch onbrunn et al., 2001; Sammons et al., 2007). In addition, multiple alterations of the same enzyme have been found for ALS and ACCase inhibitors (Tranal an d Wright, 2002). This may explai n the apparent high frequency

of resistance and the short time in which resi stance developed to herbicides in these twoMonsanto Company 09-SY-201U Page 376 of 471 classes of chemistries. Only one altered site in the targeted plant EPSPS enzyme has been found for glyphosate (Baerson et al., 2002).

Herbicide resistance as a result of exclus ion mechanisms is the glyphosate resistant mechanism among the majority of the weed species studied to date. This resistance

mechanism has also been found to be associat ed with 2,4 D and paraquat. Within this category, there are two types of translocation alterations that have been observed for glyphosate; (a) restricted moveme nt of glyphosate from leaf cells into the meri stematic cells of the plant and (b) restricted movement of glyphosate within a cell into the chloroplast due

to accumulation within the vacuol e. The level of glyphosate resi stance conferred with this mechanism is higher (6-8X) than for species exhibiting target site mutations (2-3X).

The third type of herbicide resistance mechan ism, metabolism, has not been found to be a resistance mechanism associated with glyphosate in any of the weed specie s studies thus far. However, legumes have been shown to degr ade glyphosate and theref ore this type of resistance mechanism may be active in some species (Reddy et al., 2008).

In some species the experimental evidence s uggests that multiple mechanisms of glyphosate resistance may occurr within the same plant a nd multiple mechanisms are needed to protect the plant from the phytotoxic effects of glyphos ate (Yu et al., 2007). This implies that multiple genes (polygenic resistance) are necessary and thus the selection of plants with multiple genes needed to confer resistance woul d be expected to occur at a low frequency.

In summary, the overall low occurrence of glypho sate resistance may be in part explained by: (1) the nature of the target site inhibition by glyphosate relativ e to other herbicides, (2) the

lack of metabolism as a mechanism of selectiv ity for weed resistance, and (3) evidence of multiple mechanisms being necessary for resistance and thus resistance is polygenic and

difficult to assemble and maintain.

L.6 Use of Recommended Glyphosate Rate

The interaction between herbicide application rate and resistance for postemergence herbicides, such as glyphosate, is dependent upon the nature of the plant gene(s) conferring resistance to the chemical. In general, he rbicide rate has more effect on selecting for resistant individuals in a popula tion if the resistant gene is semi-dominant or recessive as compared to the resistant gene being dominant . Likewise, herbicide rates would have more

of an effect on the onset of resistance if co mmercially significant resistance required the additive effect of multiple genes (i.e. quantit ative or polygenic resistance). Low rates would tend to allow certain biotypes to survive and ma te with other biotypes of the same or an alternate resistant gene. The offs pring of this mating may then be able to survive a full rate.

Less-than-recommended or suboptimal rates have been implicated or speculated as the causal

factor in herbicide resistance for several differe nt herbicides, including chlortoluron-resistant blackgrass, diclofop-resistant ryegrass and dicamba-resistan t kochia (Beckie, 2006). Busi (2009) demonstrated that, in thre e generations of a ryegrass biot ype sprayed at sublethal rates of diclofop-methyl or glyphosate, a high level of resistance evol ved to diclofop-methyl and a moderate level to glyph osate. The conclusion of this wo rk was that growers should avoid lowering the application rate of herbicides, especially where major cross-pollinating weed species, such as lolium, are present.Monsanto Company 09-SY-201U Page 377 of 471 L.7 Weeds Resistant to Glyphosate

As with any other herbicide, the use of glyphosate may l ead to the development of glyphosate-resistant weed species , and a list of glyphosate resist ant weeds is provided below in Table L-1. However, the potential for th e development of a glyphosate-resistant weed needs to be considered in the following c ontext: (1) if a glyphosat e-based weed control system were not available, other herbicide(s) with equal or greater potential for resistance would be used to control weeds and (2) other he rbicides and cultural practices can be used to manage the glyphosate resistant specie s (Neve, 2008; Gustafson, 2008).

To date, biotypes of fifteen weed species resi stant to glyphosate have been identified and confirmed worldwide. Nine species resistant to glyphosate have been confirmed in the U.S.,

two of which were identifie d outside of Roundup Ready croppi ng systems. The speed of spread and geographical distribu tion of the resistant species has varied. Some species with resistant biotypes, such as common ragweed ( Ambrosia artemisfolia ) have been found in a limited number of sites across the mid-west, whereas marestail ( Conyza cana densis) has been found in many states in the northeast, mid-west and the south. The reproductive biology of the particular weed sp ecies involved appears to be a fact or contributing to the spread of resistant biotypes. In the above examples, marestail produces a large number of winddispersed seeds, which contribu tes to rapid spread, while ragw eed seeds do not have features that allow for such easy distribution by the wind (Weaver, 2001; Pennsylvania State University Cooperative Extension Servic e Weed Identification Bulletin #8, 1986).

Table L-1. U.S. Glyphosate Resistant Weeds through June 2009

Weeds identified outside of Roundup Ready Systems Rigid ryegrass ( Lolium rigidum )

Hairy fleabane ( Conyza bonariensis )

Weeds identified in Roundup Ready Systems Horseweed ( Conyza canadensis ) Common ragweed ( Ambrosia artemisiifolia ) Giant ragweed ( Ambrosia trifida ) Palmer amaranth ( Amaranthus palmeri ) Common waterhemp ( Amaranthus rudis ) Italian ryegrass ( Lolium multiflorum ) Johnson grass ( Sorghum halepense )Monsanto Company 09-SY-201U Page 378 of 471 Some weed species, such as Equisetum arvensi s (field horseweed), are tolerant, as opposed to resistant, to herbicides. In addition, some species are more diffi cult to control with glyphosate than others (e.g. lambsquarters ( Chenopodium album ) and morninglory ( Ipomea sp.) and require more care to make sure the co rrect amount of glyphosate is applied at the right growth stage. For these difficult-to-contr ol weeds, environmenta l conditions can affect herbicide performance more than for weeds that are easier to c ontrol and therefore it is more critical that the correct rate be applied at th e right growth stage when making applications to

weeds in the difficult-t o-control category. Weed control situations involving tolerant or difficult-to-control species are of ten confused with resistance.

L.8 Use of Glyphosate for In-crop Weed Management

Monsanto has developed plants through biotec hnology to be tolerant to glyphosate. The development, approval and cultivation of these Roundup Ready crops have facilitated additional uses of glyphosate in crops where such uses were not previously possible given the non-selective nature of glyphosate. This development has provide d growers with an additional weed management option and benef its relative to existing weed management options. The glyphosate-tolerant trait in Roundup Ready crops has no effect per se on the

control of weeds. From a weed resistance st andpoint, the use of gl yphosate with glyphosatetolerant soybean is no different than the us e of a selective herbicide in a conventional soybean crop.

The most often cited benefits of glyphosate as an in-crop weed management option are simplicity, flexibility of application timing, weed spectrum, crop safety, and environmental safety (Dill, 2005). The ability to use glyphosate in-crop has a llowed farmers to change their farming practices in some cases. For ex ample, Roundup Ready cotton and Roundup Ready soybean have often been cited as a major reas on for an increase in no-till practices (Dill et al., 2008).

Since Monsanto commercialized the first Roundup Ready soybean variety in 1996, growers have enthusiastically adopted the technology. Currently, bi otechnology-derived herbicide tolerant soybean is planted on over 92% of U. S. soybean acreage (U SDA-NASS, 2008). The Roundup Ready soybean system, (i.e., plan ting Roundup Ready soybean and applying glyphosate in-crop), has become the standard weed control program in U.S. soybean production. In addition, w eed control in a Roundup Ready soybean system likely will involve not only glyphos ate-based herbicides but also other herbicides and weed management practices to effectively manage weeds, thus increasing crop yield and reducing development of resistant weed s. State Universities/Coopera tive Extension Services (CES) publish information on best weed management practices in Roundup Ready crops to address both of these objectives (see Table L-2). In addition Monsanto and other companies selling glyphosate products provide information on thes e same best management practices as detailed later in this Appendix.

L.9 Weed Resistance Management Strategies for Glyphosate

As part of Monsanto’s stew ardship of Roundup agricultura l herbicides and Roundup Ready crop systems, the company has conducted inve stigations and worked extensively withMonsanto Company 09-SY-201U Page 379 of 471 academics and other herbicide manufactures to understand the best practices to manage resistance. These investigations have demonstrated that one of the major factors contributing to the development of resistant weeds is poo r weed control manageme nt practices. These include application of herbicid es at rates below t hose indicated on th e EPA-approved label for the weed species and sole reliance on a part icular herbicide for w eed control without the

use of other herbicides or cult ural control methods (i.e. pre-pl ant and in-crop tillage) (Beckie, 2006; Peterson et al., 2007).

The weed resistance manageme nt recommendations that will be made for the use of glyphosate in conjunction with va rieties developed from MON 87705 will be consistent with the Herbicide Resistance Action Committee’s gu idelines for prevention and management of herbicide resistance (H RAC, 2009). These guidelines recomme nd an integrated approach to weed resistance management including crop ma nagement (i.e. row spacings, etc), cultural techniques and herbicides.

EPA is the U.S. federal regulatory agency that administers the federal law governing pesticide sale and use (FIFRA). EPA encourag es pesticide manufacturers to provide growers with information regard ing a herbicide’s mode of action to aid growers in planning herbicide use practices and to foster the adoption of ef fective weed-resistance management practices as specified by EPA in PR Notice 2001-5. In that document EPA states that “this approach to resistance management is sound and would be highly beneficial to pesticide manufacturers and pesticide users” (EPA PR Notice 2001-5 at http://www.epa.gov/opppms d1/PR_Notices/pr2001-5.pdf ). EPA approves all pesticide label use instructions based on the agency’s evalua tion of supporting data supplied by the pesticide registrant or manufacturer. Afte r EPA approves a pesticide label, it is a violation of federal law to use the pesticide for a use or in a manne r not in accordance with the label directions.

Monsanto incorporates EPA’s guidelines for pesticide resist ance management labeling on its glyphosate-based agricultural herbicide labels, a nd will do so on the label for products to be applied over the top of varieties devel oped from MON 87705 (An example of current Roundup WeatherMAX product la bel is available at www. cdms.net/ldat/ld5UJ029.pdf ). EPA-approved labels for Roundup branded he rbicide weed -resistant management recommendations are designed to minimize th e potential for the development of glyphosateresistant weeds. By approving a label for a gl yphosate-based agricultura l herbicide, EPA has concluded that the product will not cause unreasonable adverse effects to the environment or

human health when used in accordance with the label’s directions.

The weed resistance management guidelines on the labels of Roundup ag ricultural herbicides include recommendations that are well-documented in the scientific literature as being appropriate and effective for weed control and to mitigate weed resistance. Significant research has been conducted to identify the appropriate application rate of glyphosate required to control a particular weed at various growth stag es under various agronomic and

environmental conditions. These rates are based on over 35 years of ongoing research at Monsanto to evaluate the efficacy of Roundup ag ricultural herbicides. Studies have included efficacy of weed control for a broad spectrum of weeds and under a wide range of conditions.

Research has also involved th e study of tolerance of the Roundup Ready crops to over-thetop applications of Roundup agricultural herbicid es. Delaying herbicide application allows weeds to grow larger, requiring higher herbic ide rates for control. At lower glyphosate application rates, larger w eeds are controlled less cons istently (Gubbiga et al., 2002). A keyMonsanto Company 09-SY-201U Page 380 of 471 element of effective weed control and weed re sistance management, therefore, is using the correct rate of glyphosate at the right time for the weed species and the size of the weed (i.e., using a lethal dose which avoids the need for subsequent applications). This important strategy is well-supported by fiel d research studies at several universities (Wilson et al., 2003; Jeschke et al., 2006; Stolte nberg, 2002; Wilson et al., 2006). Additionally, it is accepted in the weed science community that th e use of multiple herbicide modes of action via tank mixtures, use of herbicides with diffe rent modes of action in a rotational crop, or using multiple herbicides in sequence within a crop will reduce the risk of developing weed resistance (Gessel and Stegal, 1990; Beckie, 2006). Tank-mixing involves mixing two or more herbicides in the spray tank immediately prior to application. Simultaneously using two herbicides with different modes of action significantly reduces the probability of weeds developing resistance to either or both chemistries. To pr ovide growers with the tools needed to minimize resistant weed development, Monsanto will continue to investigate and recommend appropriate residual and postemergence herbicide produc ts that have a different mode of action from glyphosate. As an example, the herbicide metolachlor (tradename PARRLAYTM) is a residual herbic ide that will help reduce flushes of annual grasses and pigweed which could slow the selection and pote ntial spread of glyphosat e-resistant weeds in Roundup Ready cotton and Roundup R eady Flex cotton systems.

Crop rotation and management of the fallow peri od and cover crops, has been found to be an important consideration in managing resistance . In general, crop ro tation fosters use of alternate herbicide modes of action and, poten tially, use of additional cultural practices to manage weeds over time (Beckie et al., 2004). Several authors have referred to this general concept as applying “diversity” across croppin g/fallow seasons to manage weed resistance (Beckie, 2006; Powles, 2008). In general, cons ervation tillage practi ces (minimum-till and no-till) are viewed as creating environments where herbicide resistance is likely to develop. This is probably due to heavy selection pr essure put on weeds by herbicides in these environments and the absence of tillage as a cultural practice to supplement herbicides. However, this is not always the case. Legere et al. (2000) found that an increase in ACCase inhibitors (aryloxyphenosy propionate and cyclohexanedinone chemical families) use in conservation tillage system did not result in an increased incidence of wild oat populations resistant to ACCase inhibitors. In addition, preplant and/or in-crop cultivation will actually promote the spread of some species such as Johnsongrass. Therefor e, no-till would be a better cropping system to contain Johnsongrass populations that could contain a resistant gene (McWhorter, 1972).

The stewardship program for managing w eed resistance in MON 87705 soybean is essentially the same program that is alrea dy in place for Roundup Ready soybean event 40-3- 2 and for Roundup Ready 2 Yield soybean. Th e weed-resistance management guidelines used for varieties developed from MON 87705 will be consistent with those that growers of Monsanto’s products are cont ractually obligated to ab ide by through the Monsanto Technology Stewardship Agreement (MTSA). Each soybean grower who will purchase a variety developed fr om MON 87705, or any other va riety containing the Roundup Ready trait, must enter into a limite d use license with Monsanto and must sign and comply with the MTSA. The MTSA incorporates by reference an d requires the grower to follow Monsanto’s Technology Use Guide (TUG), which sets forth th e requirements and best practices for the cultivation of crops with the Roundup Ready tr ait including recommendations on weedresistance management practi ces. Among each grower’s legal commitments is the federalMonsanto Company 09-SY-201U Page 381 of 471 requirement to use all Roundup herbicides in acc ordance with the directions for use on their labels, which incorporate weed resistance manage ment practices. These are inclusive of the weed resistance management guidelines that are found on the EPA approved Roundup agricultural herbicide labels.

The weed-resistance manage ment practices articulated in the TUG are broadly communicated to growers and retailers in order to minimize th e potential for the development of glyphosate-resistant weeds. These practices are communicated through a variety of means, including direct mailings to each grower purchasing a Roundup Ready crop product, a public website (www. weedresistancemanagement.com), and reports in farm media publications. The overall stewardship program is reinforced through collaborations with U.S. academics who provide their recommendations fo r appropriate stewardship of the use of Roundup agricultural herbicides with Roundup Ready crops, as well as by crop commodity groups who have launched seve ral weed-resistance educationa l modules available on their websites. Finally, Monsanto urges farmer s to report any incidence of repeated nonperformance of Roundup agricultu ral herbicides on a particular weed, and Monsanto investigates cases of unsatisfactory weed contro l to determine the cause as defined in Section L-10 of this Appendix.

In cases where resistance is confirmed, Monsanto a nd University/CES provide

recommendations for alternat ive control methods for farm ers (see Table L-2). These recommendations are made available through Monsanto supplemental labels, Monsanto TUG, Monsanto and University publications a nd internet sites to gr owers, consultants,

retailers and distributors. In all cases of glyphosate-resistant weeds in the U.S. and globally,

there are alternative he rbicides and cultural methods available to farmers to effectively control these species. Some examples of these recommendations from University/CES personnel are found in Table L-2. It is importa nt to note that ther e are many alternative options in each situation.Monsanto Company 09-SY-201UPage 382 of 471Table L-2. Management Reccomendations for Control of Glyphosate Resistant WeedsGlyphosateResistantWeedState Crop Recommendations for alternative herbicides tomanage glyphosate resistant weeds1Reference(Bulletin No.)PalmeramaranthAR Soybean Burndown: flumioxazin andPre: flumioxazin or metolachlor, and/orPost: fomesafinU of AR (FSA2152)www.uaex.eduAR Cotton PPI: triflualin or pendimethalin and/orPre: diuron or fluometuron and/orE. Post: metolachlor and/orPost directed: diuron or prometryn orLayby: flumioxazinU of AR (FSA2152)www.uaex.eduWaterhemp MO Corn Pre: metolachlor or acetachlor or isoxaflutole ormesotrione or atrazine, and/orPost: atrazine or dicamba or 2,4DU of MO (IPM1030)www.extension.missouri.eduMO Soybean Pre: metribuzine orsulfentrazone or metolachlor orflumioxazin and/orPost: lactofen or fomesafen or aciflorfenU of MO (IPM1030)www.extension.missouri.eduCommonragweedOH /INCorn Pre: atrazine or dicamba or acetochlor and/orPost: dicamba or tembotrione or mesotrione, ortroprmezone2009 OH/IN Weed Control Guide(789)www.btny.purdue.edu/weedscienceOH /INSoybean Burndown: 2,4D andPre: metribuzine or flumioxazin or cloransulam and/orPost: cloransulam or fomesafen or lactofen2009 OH/IN Weed Control Guide(789)www.btny.purdue.edu/weedscienceGiantragweedOH /INCorn Burndown: 2,4D +atrazine andPre: Lumax or atrazine+isoxaflutole and/orPost: atrazine or dicamba or tembotrione or mesotrione,or troprmezone2009 OH/IN Weed Control Guide(789)www.btny.purdue.edu/weedscienceMonsanto Company 09-SY-201UPage 383 of 471Table L-2 (cont.). Management Reccomendationsfor Control of Glyphosate Resistant WeedsGlyphosateResistantWeedStateCropRecommendations for alternative herbicides tomanage glyphosate resistant weeds1Reference(Bulletin No.)GiantragweedOH /INSoybean Burndown: 2,4D andPre: Canopy or Envive orimazaquin or Authority orflumioxazin or cloransulam and/orPost: cloransulam or fomesafen or lactofen+bentazon2009 OH/IN Weed Control Guide(789)www.btny.purdue.edu/weedscienceMarestail TN Corn Burndown: 2,4D or dicamba orPre: atrazine and/orPost: dicamba2009 TN Weed Control Guide(PB1580)www.weeds.utk.eduSoybean Burndown: 2,4D ordicamba or flumioxizin orPre: metribuzin or fluioxazin and/orPost:cloransulam2009 TN Weed Control Guide(PB1580)www.weeds.utk.eduCotton Burndown: dicamba orflumioxazin or trifloxysulfuronorPre: fluometuron or diron or prometryn and/orPost:trifloxysulfuron and/orPost-directed: flometuron+MSMA or diuron+MSMA orprometryn+trifloxysulfuron2009 TN Weed Control Guide(PB1580)www.weeds.utk.edu1(Burndown=before planting; Pre= preemergence;Post= postemergence; Post-directed= applied postemergence directed at the base ofthe crop; PPI=Pre Plant Incorporated)Monsanto Company 09-SY-201U Page 384 of 471 L.10 Monsanto Weed Performance Evaluati on and Weed Resistance Management Plan

To support and enhance Monsanto ’s weed management princi ples, stewardship program, and grower recommendations, Monsanto implements a Weed Performance Evaluation Program (WPEP) based on grower performance inquiries and field tr ial observations. In addition, a Monsanto Resistance Management Plan (MRMP) will be implemented as

warranted. Each of these is discussed below.

The goal of the WPEP program is to continue to adapt, modify, and improve Monsanto’s

weed control recommendations to growers, with a focus on : (a) identifying performance issues with particular weeds and growi ng conditions; (b) providi ng product support to growers who are not satisfied with their leve l of weed control; and (c) identifying and investigating potential cases of glyphosate resistan ce early so that mitigation strategies can be implemented.

The WPEP is initiated when a grower reports to Monsanto, a retailer and/or University/CES personnel instances of unsatisfactory weed c ontrol following a glyphos ate application. Follow-up with the grower is initiated by any of the three parties listed above to understand the situation, understand the re ason for the inquiry and resolve it to the satisfaction of the customer. By virtue of Monsanto’s presence in the marketplace, in cases where the initial contact with the grower is a retailer or Univ ersity/CES person, Monsanto will be contacted and will engage in the technical investigation. It is important to Monsanto, as part of its proactive stewardship commitment, that thes e product performance inquiries are acted upon and investigated quickly.

The vast majority of these reported herbicide pe rformance issues are determined to be due to application error or environmen tal conditions and ar e resolved through a phone conversation with the grower. However, a system is in place to investigate isolated or recurring performance problems for a weed species in a specific field. As warranted, a Monsanto, retailer or University/CES technical representative will arrange an on-site visit. If resistance is suspected to be a cause of the performan ce problem and has not already been confirmed for the specific weed and/or geography, additiona l steps will be taken to further explore the possibility of a new resistant weed species or population. Th ese are the first steps of the Monsanto Resistance Management Plan (MRM P). When the reporte d problem involves a weed where resistance is common in the geogra phy, the technical representative may simply confirm that the grower is managing a resist ant population in an appropriate manner.

The MRMP consists of two primary elements: (a) initiation of testing steps to verify cases of suspected resistance, and (b) development and communication of Monsanto and/or University/CES guidelines to incorporate re sistance mitigation into weed management programs where resistance is confirmed.

As part of the steps to verify cases of su spected resistance and if the on-site technical investigation cannot rule out poten tial resistance, seed may be co llected from the subject site for follow-up greenhouse trials at Monsanto or at another University or third-party testing facilities that are set up to do th is type of evaluation. If gr eenhouse trials do not repeat the observation and the weed is clearly controlled at label rates, then a thorough follow-up visit is conducted with the grower to re-evaluate the application recommendations and conditions of the operation that may be impacting weed c ontrol. If the greenhouse efficacy trials doMonsanto Company 09-SY-201U Page 385 of 471 indicate insufficient control at label rates, th en additional studies may be conducted to verify if the weed is resistant or, if the Monsanto, University/CES or third-party researcher has sufficient evidence, resistance may be considered to be confirmed wit hout further testing.

Resistance is considered to be confirmed if the two resistance criteria outlined in the Weed Science Society of America (www.wssa.net ) (described in section L.3) are deemed to be fulfilled either through field and/or greenhouse data.

When resistance is confirmed, the scientific and grower communities are notified and a weed resistance mitigation plan is implemente d by Monsanto in cooperation with the University/CES. The mitigation plan is design ed to manage the resistant biotype through effective and economical weed management recommendations implemen ted by the grower. The scope and level of intensity of the mitigat ion plan may vary depending on a combination of the following factors: (a) bi ology and field characteristics of the weed (seed shed, seed dormancy, etc.), (b) importance of the weed in the agricultural system, (c) resistance status of

the weed to other herbicides with alternate mode s of action, and (d) availability of alternative control options. These factors are analyzed by Monsanto and Univer sity/CES personnel in combination with economic and practical manage ment considerations to develop a tailored mitigation strategy. The plan considers what is technically appropriate for the particular weed and incorporates practi cal management strategies th at can be implemented by the

grower.

After a mitigation plan is developed, Mons anto communicates the plan to the grower community through the use of supplemental labeling (labeling which includes newly approved uses, use directions, or other instruct ions which have been added since the last EPA-approved Master label), info rmational fact sheets, retailer training program s, agriculture media and/or other means, as appropriate.

The final step of the MRMP may include ex tensive genetic, biochemical or physiological analyses of confirmed cases of glyphosate re sistant weeds in orde r to elucidate the

mechanism of resistance. The research fi ndings are communicated to the scientific community through meetings and publications , and information pertinent to field applications is incorporated into weed management recommendations.

In addition to the grower inquiry initiated proc ess, Monsanto, alone an d in cooperation with University/CES, conducts field studies to unde rstand the potential for weed resistance and

weed shifts as the result of various weed management pr ograms implemented in a Roundup Ready crop. These studies allow researchers to better track speci fic factors that can influence the development of resistance to specific weeds.

L.11 Summary

Development of weed resistance is a complex process that can be difficult to accurately predict. No single agronomic practice will mitigate resistance for all herbicides or all weeds. As a result, weed resistance needs to be mana ged on a case-by-case basis and tailored for the particular herbicide and weed in order to meet grower needs. Using good weed management principles, built upon achieving hi gh levels of contro l through proper app lication rate, choice of cultural practices, and appropriate comp anion weed control tools will allow Roundup agricultural herbicid es to continue to be used effectively. In cases where weed populations have developed resistan ce to glyphosate, effective management options are available andMonsanto Company 09-SY-201U Page 386 of 471 experience has shown that growers continue to find value in using glyphosate in their weed control programs.

The key principles for effective stewards hip of glyphosate use, including Roundup Ready crops, include: (1) basing weed management and weed resistance management practices on local needs and using the tools necessary to optimize crop yield, (2) using proper rate and timing of application, (3) not relying solely on one herbicide weed control option across a cropping system, (4) responding rapi dly to instances of unsatisf actory weed control, and (5) providing up to date weed management a nd weed resistance ma nagement training.Monsanto Company 09-SY-201U Page 387 of 471 Appendix L References

Baerson, S.R., D.J. Rodriguez, M. Tran, Y.M. Feng, N.A. Biest, and G.M. Dill. 2002. Glyphosate-resistant goosegrass. Identification of a mutati on in the target enzyme 5- enolpyruvylshikimate-3 phosphate synt hase. Plant Physiology 129:1265-1275.

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed Technology 20:793-814.

Beckie, H.J., L.M. Hall, S. Meers, J.J. Laso, and F.C. Stevenson. 2004. Management practices influencing herbicide resistan ce in wild oat. Weed Technology 18:853-859.

Busi, R., S. M. Velayudhan, and S.B. Powles . 2009. Use of below label herbicide rates can

lead to evolution of herbicide resistant weeds. Abstract No. 296. Proceedings of the Weed Science Society of America 2009.

Cole, D.J. 1985. Mode of action of glyphos ate-a literature anal ysis. The herbicide glyphosate:48-74.

Dalley, C.D., K.A. Renner, and J.J. Kells. 2001. Weed competition in Roundup Ready soybean and corn. Michigan State University, Dept of Crop and Soil Science.

Dill, G.M., C.A. Jacob, and S.R. Padgette. 2008. Glyphosate resistant crops: Adoption, use and future considerations. Pest Management Science 64:326-331.

Dill, G.M. 2005. Glyphosate-resistant crops: Hist ory, status and future. Pest Management

Science 61:219-224.

Ferguson, Gabrielle M., Allan S. Hamill, and François J. Tardif. 2001. ALS inhibitor resistance in populations of Powell amaranth and redroot pigweed. Weed Science 49:448-453.

Fernandez-Cornejo, J. and W.D. McBrid e. 2002. Adoption of bioengineered crops. Agricultural Economic Report AE R810, U.S. Department of Ag riculture, Washington, D.C.

Franz, J., M.K. Mao, and J.A. Sikorski. 1997. Glyphosate: a Unique Global Herbicide. American Chemical Society, Washington, D.C.

Giesy, J.P., S. Dobson, and K.R. Solomon. 2000. Ecotoxicological risk assessment for Roundup herbicide. Rev. Environ. Contam. Toxicol. 167:35-120.

Gustafson, D.I. 2008. Sustainable use of gl yphosate in North American cropping systems. Pest Management Science 64:409-416.

Heap, I. 2009. Internatio nal survey of herbicide resistant weeds. http://www.weedscience.org/in.asp [Accessed July 1, 2009].

HRAC. 2009. Classification of herbicides accord ing to mode of action. Herbicide Resistance Action Committee. http://www.hracgl obal.com/ [Accessed July 1, 2009].Monsanto Company 09-SY-201U Page 388 of 471 Jeschke, M.R. and D.E. Stoltenberg. 2006. W eed community composition over eight years of continuous glyphosate use in a corn-soybean rota tion. Pages 33-34 in Weed Science Society of America Proceedings.

Lee, L.J. and J. Ngim. 2000. A first repor t of glyphosate-resist ant goosegrass (Eleusine indica L. Gaertn) in Malaysia. Pe st Management Sc ience 56:336-339.

Legere, A., H.J. Beckie, F.C. Stevenson, a nd A.G. Thomas. 2000. Survey of management practices affecting the occurre nce of wild oat (A vena fatua) resist ant to acetyl-coA carboxylase inhibitors. W eed Technology 14:366-376.

Lorraine-Colwill, D.F., T.R. Hawkes, P.H. W illiams, S.A.J. Warner, P.B. Sutton, S.B. Powles, and C. Preston. 1999. Re sistance to glyphosate in Loliu m rigidum. Pesticide Science

55:489-491.

McWhorter, C.G. 1972. Factors affecting johnsongrass rhizom e production and germination.

Weed Science 20:41-45.

Neve, P. 2008. Simulation modeling to unders tand the evolution and management of glyphosate resistance in weeds. Pest Management Science 64:392-401.

Peterson, D., B. Olson, K. Al-Khatib, R. Currie, J.A. Dille, J. Falk, P. Geier, D. Regehr, P. Stahlman, C. Thompson. 2007. Glyphosate st ewardship. Kansas State University Agricultural Expewriment Station and Coope rative Extension Serv ice Bulletin MF2767.

Powles, S.B. 2008. Evolved glyphosate-resistan t weeds around the world: Lessons to be learnt. Pest Manageme nt Science 64:360-365.

Reddy, K.N., A.M. Rimando, S.O. Duke, a nd V.K. Nandula. 2008. Aminomethylphosphonic acid accumulation in plant species treated with glyphosate. Journal of Agricultural and Food Chemistry 56:2125-2130.

Ross, M.A. and C.A. Lembi. 1985. Applied Weed Science. Burgess Publishing Co., Minneapolis.

Sammons, R.D., D.C. Heering, N. DiNicola, H. Glick, and G.A. Elmore. 2007. Sustainability and stewardship of glyphosate and glyphosate- resistant crops. Weed Technology 21(2):347-

354.

Schonbrunn, E., S. Eschenburg, W.A. Shuttleworth, J.V. Schloss, N. Amrhein, J.N.S. Evans,

and W. Kabsch. 2001. Interaction of the herb icide glyphosate with its target enzyme EPSP synthase in atomic detail. Proceedings of the National Academy of Sciences U.S.A. 98:1376-

1380.

Stoltenberg, D.E. 2002. Weed management and agronomic risks associ ated with glyphosateresistant corn and soybean cropping systems. Proceedings Fertilizer Aglime and Pest Management Conference Cooperative Extension Service, University of Wisconsin, Madison

41:200-208.Monsanto Company 09-SY-201U Page 389 of 471 Tranel, P.J. and T.R. Wright. 2002. Resistance of weeds to AL S-inhibiting herbicides: What

have we learned? Weed Science 50:700-712.

USDA-NASS. 2007. Acreage 2007 (J une report). U.S. Department of Agriculture National Agricultural Statistics Service, Washington, D.C.

Weaver, S.E. 2001. The biology of Canadian weeds. 115. Conyza Canadensis. Canadian

Journal of Plant Science 81:867-875.

Wilcut, J.W., R.M. Hayes, R.L. Nichols, S.B. Clawis, J. Summerlin, D.K. Miller, A. Kendig, J.M. Chandler, D.C. Bridges, B. Bracke, C.E. Snipes, and S.M. Brown. 2003. Weed management in transgenic cotton. North Caro lina State University Technical Bulletin 319.

Williams, G.M., R. Kroes, and I. Munro. 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, gl yphosate for humans. Regulatory Toxicology and Pharmacology 31:117-165.

Wilson, R.G., S. Miller, P. Weestra, and P. Stahlman. 2006. Sustainability of glyphosate resistant cropping systems. Page 60 in Weed Science Society of America Proceedings.

Wilson, R. and P. Stahlman. 2003. Maintain th e rate. Pages 12-13 in Nebraska Farmer, June

2003.

Yu, Q., A. Cairns, and S. Powles. 2007. Glyphos ate, paraquat and ACCase multiple herbicide

resistance evolved in a Lolium ri gidum biotype. Pl anta 225:499-513.Monsanto Company 09-SY-201U Page 390 of 471 Appendix M. Nutritional Impact Ev aluation of MON 87705 Soybean Oil

M.1 Intended Food Uses of MON 87705 Soybean Oil

It is anticipated that MON 87705 soybean oil would substitute for the liquid soybean oil in the market place. The use of liquid soybean oi l is mainly in salad dressings, mayonnaise and spreads, home use liquid soybean oil, and margar ines (stick, tub, light variety). These four food categories represent approximately 94% (5,498 million pounds) of the replaceable liquid soybean oil market in the U.S. (HyQuest, 2009).

In order to assess the nutritional impact from the introduction of MON 87705 into the marketplace, a dietary replacemen t model was used. In this model, MON 87705 soybean oil was substituted for the liquid soybean oil fracti on in a subset of foods in the U.S. diet, including salad dressings, mayonnaise and spreads, home use liquid soybean oil, and margarines. This assessment assumed that all of the targeted oil components of the foods proposed for replacement at all eating occasions in the U.S. are replaced with MON 87705 soybean oil. The results presented in this a ssessment represent a c onservative estimation of the effect of MON 87705 soybean oil on fatty acid (FA) composition of the diet. The

assessment is exclusive of fr ying applications, which genera lly utilize hydrogenated or low linolenic oils. Fatty acid intake estimates in this dietary replacement model were prepared for Monsanto by Exponent (Washington D.C.)

A baseline FA composition for the soybean oil component of the four target food categories (mentioned above) was created using a combina tion of published FA compositions of liquid and partially hydrogenated soybean oils (IS EO, 2006; Exler et al., 1993), and market research analysis on currently utilized oil blends for the target foods.

M.2 Estimated Dietary Intake of MON 8770 5 Soybean Oil based on Proposed Food

Uses

Four major sources of data we re used to conduct the analys is: (1) Food intake data and nutrient composition from National Health and Nutrition Examination Survey (NHANES, CDC, 2007) 2003-2004 and 2005-2006, (2) Target foods and associated oils and Exponent’s proprietary recipes, (3) Baseline fatty acid profile for the oils associated with target foods, and (4) Fatty acid profile for MON 87705 soybean oil. Exponent’s proprietary software package, FARETM version 8.43 was used to implement the analysis. The fatty acid composition of MON 87705 used in the dietary replacement model is summarized in Table

M-1.Monsanto Company 09-SY-201U Page 391 of 471 Table M-1. Fatty Acid Profile for MON 87705 Used in the Intake Estimations

Fatty Acids Composition (% wt of Total FA)

16:0 2.4

18:0 3.3

18:1 76.5

18:2 10.1

18:3 6.7

Substitutions of MON 87705 soybean oil were done at the 100% substitution level, i.e

., assuming 100% of the liquid soybean oil contai ned in these foods wa s replaced with the MON 87705 soybean oil, and every eating occas ion of a target food was replaced by a comparable food containing MON 87705 soybean oil.

M.3 Estimated Daily Intake of Total Fat and Selected Fatty Acids before and after Replacement of Conventional Soybean Oil with MON 87705 Soybean Oil in Targeted

Foods

The mean and 90 th percentile intakes of total fat and five selected FA from conventional soybean (prior to substitution with MON 87705 soybean oil) we re estimated fo r the target foods, and are reported in Tabl es M-2 and M-3. For the U. S.population, the soybean oil component of the target foods c ontributes an average of 4.27 g/da y per capita of total fat; this amounts to approximately 5.4% of total fat from the whole diet. At the 90 th percentile, per user, the total fat contribution from the soybean oil component of the target foods is 16.28 g/day, or approximately 12.6% of total fat from the whole di et. To normalize consumption of nutrients for the range of caloric intake across a population, perc ent of energy/caloric intake (% en) is used (IOM, 2002). Therefore, on a percent energy (% en) basis, the mean per capita contribution from the liquid soybean o il component of the target foods is 1.8% and

the 90 th percentile per us er is 6.83%.

In MON 87705 soybean oil, intake of 18:1 oleic acid is increased the most among the five fatty acids evaluated. For the U.S. population, the mean per capita inta ke of 18:1 oleic acid from the MON 87705 soybean oi l component from proposed foods increased from 1.04 g/day to 3.13 g/day (1.35 % en). The 90 th percentile per user intake of 18:1 oleic acid increased from 3.86 g/day to 12.22 g/day (Tables M-2 and M-3).Monsanto Company 09-SY-201U Page 392 of 471 Table M-2. Mean Per Capita Fatty Acid In take from Target Foods Pre and Post
MON 87705 Replacement, U.S.population

Fatty Acids g/day % En

Pre replacement Post replacement % change Pre replacement Post replacement % change

Palmitic 16:0 0.45 0.13 -71.11 0.19 0.06 -68.42

Stearic 18:0 0.18 0.19 5.56 0.08 0.08 0.00

Oleic 18:1 1.04 3.13 200.96 0.45 1.35 200.00

Linoleic 18:2 1.93 0.39 -79.79 0.83 0.17 -79.52

Linolenic 18:3 0.25 0.26 4.00 0.11 0.11 0.00

Total Fat Intake 4.27 4.27 0.00 1.84 1.84 0.00

Table M-3. 90 th Percentile Per User Fatty Acid Intake from Target foods, Pre and Post
MON 87705 Replacement, U.S.Population

Fatty Acids g/day % En

Pre replacement Post replacement % change Pre replacement Post replacement % change

Palmitic 16:0 1.71 0.48 -71.93 0.72 0.21 -70.83

Stearic 18:0 0.66 0.7 6.06 0.27 0.31 14.81

Oleic 18:1 3.86 12.22 216.58 1.64 5.12 212.20

Linoleic 18:2 7.9 1.58 -80.00 3.26 0.66 -79.75

Linolenic 18:3 1.03 1.05 1.84 0.42 0.44 4.76

Total Fat Intake 16.28 16.28 0.000 6.83 6.83 0.00Monsanto Company 09-SY-201U Page 393 of 471 M.4 Impact of Replacement on Total Diet

The impact of oil replacement under the propos ed food uses with MON 87705 soybean oil on the total daily intake (g/day and % en) of total fat and the five major fatty acids (palmitic, stearic, oleic, linoleic, linolenic ) in the diet of the U.S. populat ion are summari zed in Tables M-4 and M-5. As expected, the replacemen t with MON 87705 soybean oil does not change the total daily fat intake, which remains at 79.58 g/day on the mean per capita basis and at 129.4 g/day in the 90 th percentile per user group.

Table M-4. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, U.S.Population
(g/day)

Fatty Acids Mean Per Capita 90 th Percentile Per User

Pre

MON 87705 (g/day) Post

MON 87705 (g/day) % change Pre

MON 87705 (g/day) Post

MON 87705 (g/day) % change

Palmitic 16:0 14.38 13.83 -3.80 23.62 21.48 -9.00

Stearic 18:0 6.96 6.98 0.30 11.75 11.84 0.80

Oleic 18:1 27.34 30.29 10.80 45.20 57.52 27.20

Linoleic 18:2 14.75 14.12 -4.30 25.66 23.08 -10.10

Linolenic 18:3 1.42 1.42 0.00 2.49 2.50 0.20

Total Fat

Intake 79.58 79.58 0.00 129.40 129.40 0.00

Table M-5. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, U.S.Population
(% en)

Fatty Acids Mean Per Capita 90 th Percentile Per User

Pre

MON 87705 (% en) Post

MON 87705 (% en) % change Pre

MON 87705 (% en) Post

MON 87705 (% en) % change

Palmitic 16:0 6.07 5.84 -3.70 8.00 7.10 -11.30

Stearic 18:0 2.91 2.91 0.00 3.94 4.03 2.40

Oleic 18:1 11.49 12.75 10.90 15.16 20.28 33.80

Linoleic 18:2 6.23 5.96 -4.40 9.22 8.05 -12.70

Linolenic 18:3 0.61 0.61 0.00 0.95 0.96 0.80

Total Fat

Intake 33.61 33.61 0.00 42.88 42.88 0.00Monsanto Company 09-SY-201U Page 394 of 471 Table M-6. Mean Per Capita and 90th Percentile Per User Fatty Acid Intake from Total
Diet, Post MON 87705, Age and Gender Groups (g/day)

Population Total

Fat Palmitic

16:0 Stearic

18:0 Oleic

18:1 Linoleic

18:2 Linolenic

18:3

U.S. Mean 79.58 13.83 6.98 30.29 14.12 1.42

90 t h percentile 129.40 21.48 11.84 57.52 23.08 2.50

Males 1-8 Mean 64.76 12.09 5.95 23.07 10.48 1.00

90 t h percentile 100.53 17.73 9.40 38.23 16.54 1.63

Males 9-19 Mean 93.45 16.95 8.54 34.47 15.87 1.46

90 t h percentile 142.14 24.38 13.45 61.62 24.93 2.48

Males 20-49 Mean 102.91 18.08 9.10 39.27 18.02 1.77

90 t h percentile 155.60 26.78 14.44 68.31 27.48 2.97

Males 50+ Mean 87.93 14.82 7.56 34.33 15.92 1.64

90 t h percentile 139.74 22.57 12.27 62.39 26.17 2.83

Females 1-8 Mean 60.85 11.36 5.62 21.84 9.80 0.97

90 t h percentile 91.46 16.47 8.71 36.82 15.75 1.55

Females 9-19 Mean 72.86 12.96 6.57 27.22 12.92 1.24

90 t h percentile 111.56 18.71 10.24 48.62 20.25 2.08

Females 20-49 Mean 73.65 12.57 6.39 28.27 13.40 1.39

90 t h percentile 114.17 18.48 10.26 52.51 20.70 2.35

Females 50+ Mean 63.30 10.43 5.38 24.76 11.80 1.31

90 t h percentile 100.54 15.75 8.75 45.77 19.21 2.29

Following replacement with MON 87705 soybean oil, there is an overall increase in the total daily intake of 18:1 oleic aci d from 27.34 g/day (11.49 % en) to 30.29 g/day (12.75 % en) on a mean per capita basis and an increase from 45 .2 g/day (15.16 % en) to 57.52 g/day (20.28 % en) at the 90 th percentile per user (Tables M-4 and M-5). These increases post-replacement are expected, since the 18:1 ol eic acid fraction in conventiona l soybean oil is much lower compared to the 76.5% oleic acid observed in M ON 87705. The pattern of total daily intake of fatty acids in sub groups stratified by age and ge nder are similar to that of the overall U.S. population, with 18:1 oleic acid having the largest increase following replacement of conventional soybean oi l with MON 87705 soybean oil (Table M-6). Th e Dietary Guidelines for Americans Technical Report (USHHS, 2005) st ated that MUFAs are not required in the diet; however, they provide a vehicle to achievi ng total fat intake recommendations within the

context of saturated fat and PU FA recommendations. Further, substitution of oleic acid for

saturated fat in the diet can reduce low de nsity lipoprotein cholesterol (LDL-C).

Total daily intakes of 16:0 palmitic acid decrea sed as a consequence of dietary replacement with MON 87705 soybean oil (Tables M-4 and M- 5). On a mean per capita basis, 16:0 palmitic acid intake decreased from 14.38 g/day (6.07 % en) to 13.83 g/day (5.84 % en), while the decrease was predictably greater in the per user 90 th percentile group, where daily intake decreased from 23.62 g/day (8.00 % en) to 21.48 g/ day (7.10 % en) (Table M-6). Total dietary

intakes of 18:0 stearic acid we re similar in pre- and post- MON 87705 replacement analysis, in both mean per capita (6.96 g/day a nd 6.98 g/day, respectively) and 90 th percentile per user groups (11.75 g/day and 11.84 g/day) , respectively. A decrease in dietary saturated fat intake,Monsanto Company 09-SY-201U Page 395 of 471 as shown here for 16:0 palmitic acid, is consistent with current dietary recommendations (Lichtenstein et al., 2006; US DA-ERS, 2005; WHO/FAO, 2003).

Linoleic acid (18:2) intake leve ls decreased by a similar rate, from 14.75 g/day (6.23 % en) to 14.12 g/day (5.96 % en) on a mean per capita basi s; and from 25.66 g/day (9.22 % en) to 23.08 g/day (8.05 % en) in the 90 th percentile per user group. An adequate intake (AI) for 18:2 linoleic acid of 17 g/day for me n (19-50 years) and 12 g/day for women (19-50 years) has been established by the Institute of Medicine (IO M, 2002). The values observed post-replacement for the respective subgroups are consistent with thes e recommendations. For example, post replacement values are 18.02 g/day (males 20- 49) and 13.40 g/day (females 20-49) (Table VII-8). Further, the population and adult s ubgroup 18:2 linoleic intakes post replacement are

still in line with U.S. Diet ary Guidelines (USHHS, 2005) a nd the Acceptable Macronutrient Distribution Range of 5- 10% energy (IOM, 2002), which is de signed to reduce risk for chronic disease. For example, the population mean in take of linoleic acid was 6.23% energy before replacement and 5.96% energy pos t-replacement (Table M-5).

There was no change in 18:3 linolenic acid after replacement with MON 87705 oil (Table M-4, M-5). An adequate intake (AI) for 18:3 linolenic acid of 1.6 g/day for men (19-50 years) and 1.1 g/day for women (19-50 years) has been established by the Institute of Medicine (IOM, 2002). The values observed both pre and post replacement for the respective subgroups are

consistent with these recommendations. For example, post replacement values are 1.77 g/day (males 20-49) and 1.39 g/day (f emales 20-49) (Table M-6).

In summary, replacement of conventional s oybean oil with MON 87705 soybean oil under the proposed food uses results in changes in the fatty acid composition in the U.S. diet that lead to higher oleic acid intake, and lower consumption of saturated fats (i.e., 16:0 palmitic) and linoleic acid, with no impact on total fat intake. Given that this assessm ent assumes that all of the targeted oil components of the foods proposed for replacement at all eating occasions in the U.S. are replaced with MON 87705 soybean oil, the results presented here represent a

theoretical maximal effect of MON 87705 on fatty acid composition of the diet. The nutritional impact of exposure to MON 87705 soyb ean oil in targeted foods under the intended

conditions of use is estimated to result in chan ges in fatty acid consump tion that are in line with current dietary guidelines for fatty acid intake (Lichtenstein et al., 2006; USDA-ERS, 2005; WHO/FAO, 2003).

M.5 Composition and Nutrition Assessment Conclusion

In conclusion, the nutritional im pact from the use of MON 87705 soybean oil in targeted foods under the intended conditions of use, is estimated to result in changes in fatty acid consumption

that are in line with current dietary guidelines for fatty acid intake. Therefore, MON 87705 is regarded to be as safe and nutritious as conventional soybean for food use.Monsanto Company 09-SY-201U Page 396 of 471 Appendix M References

CDC. 2007. National health and nutrition survey examination surv ey data. US department of health and human services (DHHS), Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS). Data sets available from: http://www.cdc.gov/nchs/about/m ajor/nhanes/datalink.htm

Exler, J., L. Lemar, and J. Smith. Fat and Fa tty Acid Content of Se lected Foods Containing Trans-Fatty Acids. U.S. Department of Agriculture, ARS. Washington, D.C. http://www.nal.usda.gov/fnic/foodcomp/Data/O ther/trans_fa.pdf [Acce ssed July 9, 2009].

Lichtenstein, A.H., L.J. Appel, M. Brand, M. Carnethon, S. Daniels, H.A. Franch, B. Franklin, P. Kris-Etherton, W.S. Harris, B. Howard, N. Kara nja, M. Lefevre, L. Rudel, F. Sacks, L. Van Horn, M. Winston, and J. Wylie-Rosett. 2006. Diet and lifestyle recommendations revision

2006. Circulation 114:82-96.

HyQuest Partners, Soybean Oil Utilization Report, 2009

IOM. 2002. Dietary fats: Total fats and fatty acids. Pages 8-57 and 422-541 in Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. Panel on Macronutrients, . Instit ute of Medicine. National Academies Press, Washington D.C.

ISEO. 2006. Foods and fats. Pages 1-37 in Food Fa ts and Oils. Institute of Shortening and Edible Oils, Washington, D.C.

USDA-ERS. 2005. Soybeans and oi l crops: Background. U.S. De partment of Agriculture, Washington, D.C.

U.S. Department of Health and Human Serv ices and U.S. Depart ment of Agriculture (USDHHS-USDA). Dietary Guidelines for Amer icans, 2005. 6th Edition, Washington, DC: U.S. Government Printing Office, January 2005.

USHHS. 2005. Dietary Guidelines for Amer icans. HHS Publication No: HHS-ODPHP-2005- 01-DGA-A. U.S. Department of Health and Human Services, Washington, D.C. www.health.gov/dietaryguideli nes [Accessed June 7, 2009].

WHO/FAO. 2003. Diet, nutrition and th e prevention of ch ronic diseases. http://whqlibdoc.who.in t/trs/WHO_TRS_916.pdf WHO technical report: 916.Monsanto Company 09-SY-201U Page 397 of 471 Appendix N. Glyphosate-Tolerant Crops: Potential Impacts on Land Use and Agricultural Practices

N.1 Introduction

Since 1994, USDA has granted nonr egulated status to 13 gl yphosate-tolerant biotechnologyderived products (Table N-1). Petitions for th ree additional glyphosate-to lerant products have been submitted to the agency, with deregulat ion decisions pending for alfalfa, corn and creeping bentgrass. The crops for which petitions have been approved or are pending include: corn, cotton, creeping bentgrass, rapeseed (can ola), soybean, and sugar beet. This document discusses the cumulative impact s on land use and agricultural pr actices from deregulation of glyphosate-tolerant crop products, considering th e existing glyphosate-tole rant crops and those potentially available in the reasonably fo reseeable future based on pending petitions.

N.2 Impact on Land Use

USDA has granted nonregulated st atus to events conveying th e glyphosate-tolerance trait in five different crops (Table N-1). These crops have been rapidly adopted by U.S. farmers and since their introduction have been grown cumu latively on more than 778 million acres. In 2007 alone, glyphosate-tolerant crops were plan ted on approximately 120 million acres in the

U.S. 11 The sections below assess the impact of the adoption of glyphosate-tolerant crops on cropping patterns.

Table N-1. Herbicide-Tolerant Crops Undergoi ng Review or Previously Deregulated by

USDA-APHIS.

Petition Crop Species Trait(s) Status

93-258-01p Soybean Glyphosate-tolerant Deregulated

95-045-01p Cotton Glyphosate-tolerant Deregulated

96-317-01p Corn Glyphosate-tolerant and

Corn Borer-resistant Deregulated

97-099-01p Corn Glyphosate-tolerant Deregulated

98-173-01p Sugar Beet Glyphosate-tolerant Deregulated

98-216-01p Canola Glyphosate-tolerant Deregulated

00-011-01p Corn Glyphosate-tolerant Deregulated

01-324-01p Canola Glyphosate-tolerant Deregulated

03-104-01p Creeping Bentgrass Glyphosate-tolerant Pending

03-323-01p Sugar Beet Glyphosate-tolerant Deregulated

04-086-01p Cotton Glyphosate-tolerant Deregulated

04-110-01 Alfalfa Glyphosate-tolerant Pending

06-178-01p Soybean Glyphosate-tolerant Deregulated

06-271-01p Soybean Glyphosate- and Acetolactate Synthase Inhibitor-tolerant Deregulated

06-332-01p Cotton Glyphosate-tolerant Deregulated

07-152-01p Corn Glyphosate- and Imidazolinone-tolerant Pending

11 Calculated from datasets found at www.ers.usda.gov/data/biotechcrops.Monsanto Company 09-SY-201U Page 398 of 471 N.3 Total Cropland Acres

The overall U.S. area planted to principal cr ops, which include corn, sorghum, oats, barley, winter wheat, rye, durum, wheat, rice, soybean, p eanuts, sunflower, cotton, dry edible beans, potatoes, canola, proso millet, and sugar beets, has remained relatively constant over the past 25 years. From 1983 to 1995, the average yearly acreage of principal crops was 328 million. This average is statistically unchanged at 326 million acres since the introduction of glyphosate-tolerant crops in 1996 12 . If fact, the total U.S. cropland acres have been relatively constant since the 1940s (Vesterby and Kr upa, 1997). Therefore, the introduction of glyphosate-tolerant crops has not resulted in a si gnificant change to the total cropland acres in

the U.S.

Following deregulation and commercial intr oduction, the acres of glyphosate-tolerant varieties/hybrids planted for each crop has st eadily increased (see Figures N-1 through N-3 below). However, rapid adoption of glyphosate- tolerant crops has not correlated with an increase in the total acres for that particular crop. Specific crop acres do vary from year-toyear, but these fluctuations occur routinely and are due to a myriad of factors based largely around grower economic returns for each crop, crop rotation practices, and government programs. The impact of glyphosate-tolerant crop deregulation or pending deregulation and adoption on crop acres is discussed below.

N.3.1 Canola

Glyphosate-tolerant canola was commercialized in the U.S. in 1999. In 2007, U.S. growers planted 1.2 million acres of ca nola (USDA-NASS, 2007) and of those acres, 0.61 million acres, or approximately 50%, cont ained the glyphosate-tolerance trait (Monsanto, 2007).

The total canola acreage since 1990 and the acreage of glyphosate-tolerant canola since commercialization in 1999 are provided in Figur e N-1. The total U.S. acreage of canola increased 200% from 367,000 acres in 1996 to 1.1 million acres in 1998, coinciding with passage of the 1996 Federal Agriculture Improvement Act. 13 This acreage peaked at 1.55

million in 2000 and has remained near 1.l million acres since 2005.

The acreage of glyphosate-tolera nt canola increased rapidly fo llowing commercial introduction

in 1999 and has remained between 50 and 70% of planted canola acres since 2001. The

dramatic fluctuation in tota l canola acreage before and afte r glyphosate-tolerant canola was

commercialized indicates that factors unrelated to the availability of the glyphosate-tolerant trait play a larger role in acres planted than the availability of the glyphosate-t olerant trait.

12 Calculated from datasets at http://usda.mannlib.corne ll.edu/usda/current/htrcp/ by comparing the total acres

from 1983 to 1995 and from 1996 to 2007.

13 The 1996 Federal Agriculture Improvement Act, 7 U.S.C. § 7201 et seq. , gave growers almost complete flexibility in selecting the crops they could plant.Monsanto Company 09-SY-201U Page 399 of 471 Figure N-1. Total Acres of Canola Relative to Acres of Glyphosate-tolerant Varieties planted. Data obtained from http://usda.mannlib.c ornell.edu/usda/current/htrcp/ for total canola acres and www.monsanto.com/pdf/i nvestors/2007/FY2007BiotechAcres.pdf for glyphosate-tolerant canola acres.

N.3.2 Corn

Glyphosate-tolerant corn was co mmercialized in the U.S. in 1996. From 1996 to 2007, glyphosate-tolerant corn hybrids were grown on 151 million cumulative acres. In 2007, U.S. growers planted 93.6 million acres of fiel d corn (USDA-NASS, 2007); 48 million acres, or 52%, contained a glyphosate-tolera nce trait (USDA- ERS, 2007).

Since its introduction, the percen tage of glyphosate-tolerant corn has increased steadily to 52%

in 2007. The total corn acreage, however, remained rela tively steady from 1996 to 2006, with a yearly average of 78 million acres (see Figure N-2), indicating that the glyphosate-tolerant trait has had little impact on overall corn acreage. In 2007, the total corn acreage was up 15%

from 2006 to 92.9 million acres. This increase was attribut ed to increased demand from ethanol producers and strong exports sales 14 . The increase in total corn acres, including an increase in glyphosate-tolerant co rn acres, resulted in fewer acr es of soybean planted in the

Corn Belt and Great Plains and fewer acres of cotton and rice planted in the Delta and Southeast regions.

14 http://www.usda.gov/nass/PU BS/TODAYRPT/acrg0607.txt Total and Glyphosate-Tolerant Canola Acres in the US

0 200 400 600 800 1000 1200 1400 1600 1800

1990

1 9 9 2 1994

1 9 9 6

1 9 9 8 2000
2 0 0 2 2004 2006

Year1000 Acres

Canola

RR CanolaMonsanto Company 09-SY-201U Page 400 of 471 Figure N-2. Total Acres of Corn and Soybean Relative to Acres of Herbicide-tolerant (HT) Varieties Planted. The varieties are predominately gl yphosate-tolerant varieties, but also include phosphinothricin -tolerant hybrids for corn . Data obtained from http://usda.mannlib.cornell.edu/ usda/current/htrcp/ for total crop acres and USDA-ERS (2007) for herbicide-tolerant crops acres.

N.3.3 Soybean

Glyphosate-tolerant soybean was commercialized in the U.S. in 1996. From 1996 to 2007, glyphosate-tolerant soybean was grown on more than 509 million cumulative acres. In 2007, growers planted 64 million acres of soybean in the U.S. (USDA-NASS, 2007); 58 million acres, or 91%, contained the glyphosate-t olerance trait (USDA-ERS, 2007).

Since its introduction, the percentage of glyphosat e-tolerant soybean has increased steadily to 91% in 2007 (Figure 2). The total acres of s oybean increased 23% (62.4 to 76.9 million acres)

from 1996 to 2006. This increase was due to hi gher global demand, but was facilitated by increased planting flexibility from: (1) the 2002 Farm Bill; (2) steadily rising yield improvements from narrow-rowed seeding prac tices; (3) a greater nu mber of 50-50 cornsoybean rotations; and (4) lowe r production costs, partly due to widespread adoption of herbicide-tolerant varieties (Ash et al., 2006). In 2007, acres of soybean were down 17% to 64.1 million compared to 2006 due to growers pl anting more corn, as discussed above. 

### Total and Herbicide-Tolerant Corn and Soybean

Acres in the U.S.

0 20,000 40,000 60,000 80,000 100,000

1 9

90 19 9 2

1 9

94 1996 19 9 8 2 0

00 20 0 2 2 0

04 20 0 6

Year1000 Acres Corn

Soybean

HT Corn

HT Soybean Corn

Soybean HT Soybean

HT Corn 

### Total and Herbicide-Tolerant Corn and Soybean

Acres in the U.S.

0 20,000 40,000 60,000 80,000 100,000

1 9

90 19 9 2

1 9

94 1996 19 9 8 2 0

00 20 0 2 2 0

04 20 0 6

Year1000 Acres Corn

Soybean

HT Corn

HT Soybean Corn

Soybean HT Soybean

HT CornMonsanto Company 09-SY-201U Page 401 of 471 Figure N-3. Total Acres of Cotton Relative to Acres of Herbicide-tole rant (HT) Varieties

Planted. The varieties are predominately glyphosat e-tolerant varieties, but also include phosphinothricin-tolerant variet ies. Data obtained from http://usda.mannlib.cornell.edu/ usda/current/htrcp/ for total crop acres and USDA-ERS (2007) for herbicide-tolerant crops acres.

N.3.4 Cotton

Glyphosate-tolerant cotton was commercialized in the U.S. in 1997. From 1997 to 2007, glyphosate-tolerant cotton was grown on more than 76 million cumulative acres. In 2007, growers planted 11 million acres of cotton in the U.S. (USDA-NASS, 2007) of which 7.7 million acres, or 70%, contained a glyphosat e-tolerance trait (USDA-ERS, 2007).

Since its introduction, the percen tage of glyphosate-tolerant cott on increased to approximately

60% in 2001 and remained near that level unt il reaching 70% in 2007 (Figure N-3). The total cotton acreage, however, remained relatively constant from 1997 to 2006 at 14.1 million +/- 1.3 million acres (Figure N-3). In 2007, the tota l cotton acres were down 28% due to a shift to corn and soybean across the Southeast U.S. 15

N.3.5 Sugar beet

Since 1990, the total U.S. sugar beet acreage ha s remained relatively constant at 1.4 million acres (USDA-NASS, 2007). In 2007, approxim ately 1.3 million acres of sugar beet were planted in the 11 largest sugar beet production states in the U. S. Glyphosate-tolerant sugar beet has only been grown on limited acres since its deregulation in 2005, but is expected to be grown on substantially more acres in 2008. Regardless, the acres forecasted by USDA for sugar beet in 2008 are 1.08 million acres 16 , down slightly from 1.3 million acres in 2007, again

due to the market factors described above.

15 http://www.usda.gov/nass/PU BS/TODAYRPT/acrg0607.txt

16 http://usda.mannlib.cornell.edu/usda/current/Acre/ Total Cotton and Herbicide-Tolerant Cotton Acres in the U.S.

0 5000 10000 15000 20000

1990 1992 1994 1996 1998 2000 2002 2004
2 006

Year1000 Acres

Cotton

HT CottonMonsanto Company 09-SY-201U Page 402 of 471 N.3.6 Bentgrass

Creeping bentgrass is widely grown on about 77,000 acres on gol f course putting greens, tees, and fairways in the cooler clim ates throughout the U.S., and excl usively for greens in the south where specialized root zone management syst ems allow survival of creeping bentgrass under summer heat conditions. After deregulation, glyphosate-tolerant cr eeping bentgrass is expected to replace conventi onal creeping bentgrass on some golf courses. The glyphosatetolerance trait does not alter the overall biology and ecology of creeping bentgrass 17 therefore, glyphosate-tolerant creeping bentgr ass is not expected to impact land use a ny differently than conventional creeping bentgrass.

N.3.7 Alfalfa

Alfalfa grown for forage is a major crop in the U.S. More than 20 million acres have been planted annually since 1950. Acreage peaked in the mid-1960s at approximately 30 million acres and declined gradually to approxima tely 21 million acres in 2007 (USDA-NASS 2007), in part due to increased use of corn silage as a forage.

Data from the previously deregulated glyphosate- tolerant crops shows that market forces and governments programs (1996 and 2002 Farm Bills), not the presence of glyphosate-tolerant crop varieties, are primarily responsible for a ny significant changes in crop acreage. Based on the adoption rates of other glyphos ate-tolerant crops, it is expected that the deregulation and commercial availability of glyphosate-tolerant alfalfa would replace a significant portion of conventional alfalfa varieties. The availability of these glyphosate-tole rant varieties provides

for more efficient weed control and thus allows growers mo re flexibility when choosing what crop varieties to plant on their farm s. This may result in a shift b ack from corn silage to alfalfa hay for animal feed; thus increasing the alfa lfa acreage to the level seen in the 1980s and 1990s. However, based on the experience w ith the other glyphos ate-tolerant crops, deregulation of glyphosate-tolerant alfalfa is expected to have little impact on the overall acreage historically seen for alfalfa or other crops.

N.3.8 Conventional and Organic Production

Since the introduction of glyphosate- tolerant crops, the total acr eage of principal crops and acreage of each specific crop have remained relatively constant, as discussed above. Therefore, the increase in acreage of glyphosat e-tolerant crops comes at the expense of crop varieties used for conventional production, although conventional varieties are still widely

available to those who choose to plant them (see Section XI.D.3). USDA data show that the acreage of organic corn and soybean has act ually increased since the introduction of glyphosate-tolerant crops in 1996 (Figure N-4). The acreage of organi c cotton has decreased from a high of 32,850 acres in 1995 to 9,537 acres in 2005, which is comparable to the acreage planted in 1993 and 1994 before the introduction of glyphosate-tolerant cotton. The decrease in acreage is attributed to increased market competition from impor ted organic cotton (USDAERS, 2006). Therefore, the use of glyphosate- tolerant varieties has replaced conventional

17 http://www.aphis.usda.gov/b rs/aphisdocs/03_10401p.pdfMonsanto Company 09-SY-201U Page 403 of 471 varieties, resulting in a change in agricultural practices related to weed control and soil tillage, as discussed below. Based on adoption rates of other glyphosate-tolerant crops, it is expected that deregulation of glyphosate-tolerant alfalf a will result in a repl acement of a significant percentage of conventional alfalf a with glyphosate-toler ant alfalfa. Less th an one percent of alfalfa hay acres were harvested in 2005 as organic (USDA-NASS, 2006). Depending on market demand, and again based on the experiences of the corn and soybean markets, a similar increase in organic alfalfa acres is possible.

Figure N-4. Percentage of Certified Organ ic Acres for Corn, Cotton, and Soybean. Data

obtained from www.ers.usda .gov/Data/organic and www.na ss.usda.gov for total acres

.

N.4 Impact on Herbicide Use and Costs

N.4.1 Canola

For a description of weed control practi ces in conventional can ola, please see the

recommendations from the North Dakota State University Extension Service 18

. For glyphosate-tolerant canola, it is recommended that glyphosate be applied at a maximum rate of 0.38 lb acid equivalent (ae)/acre (A) with no more thantwo applications to glyphosatetolerant canola from emergence to bolting (Zo llinger, 2008). On aver age, a typical weed management program in conventional canola (tha t could provide control comparable to the program in herbicide-tolerant canola) costabout $39/A in 2005. In contrast, weedmanagement costs in glyphosate-tolerant canola, inclusive of technolo gy fee/seed premium, were about $24/A, for a total co st reduction of 62%. The net im pact on gross margins has been
an increase of between $47/ha and $55/ha for glyphosate-tolerant canola (Brooks and Barfoot, 2006).

In 2005 in North Dakota, which contained 92% of the total U. S. canola acreage, glyphosatetolerant canola was planted on 676,000 acres. Use of the glyphosate-tolerant varieties resulted

18 http://www.ndsu.edu/weeds/weed_control_guides/2009_weed_control_guide/ Percentage of Certified Organic Acres

0 0.05

0.1 0.15

0.2 0.25

1995 1997 2000 2001 2002 2003 2004 2005

YearPercent of Crop Acres

Corn

Soybean

CottonMonsanto Company 09-SY-201U Page 404 of 471 in reduction in total weed management costs by $10 million and a reduction in herbicide use by 426,000 total pounds (Sankula, 2006).

Therefore, glyphosate-tolerant canola provides significant cost savings to the growers and

reduces the amount of herbicide used.

N.4.2 Corn

A description of weed control practices in conven tional corn can be found in USDA petition 04-125-01p, p. 128 19

. A mixture of the herbicides registered for use in corn provides fair to ex cellent control of most

weeds in a conventional weed control system. However, gr owers must know the specific weed problems present on their farms or fields to select the most effective herbicide program. Typical weed management in conventional co rn includes a preeme rgence application of metalachlor+atrazine followed by a postemergence a pplication of mesotrione plus a premix of

nicosulfuron and rimsul furon (Sankula, 2006).

Two major options are used for weed management in glyphosate-tolerant corn (Sankula, 2006). The first and most widely used option is the use of half-rate of metolachlor + atrazine as a preemergence herbicide, followed by glyphosate as a postemergence application. The second approach involves a total postemergence-based progr am with either one or two applications of glyphosate or tank-mix applications of glyphosate with atrazine.

Sankula (2006) compared weed management pr ograms and costs associated with glyphosatetolerant and conventional corn . Weed management costs in 2005 were 25% lower in glyphosate-tolerant corn compared to conven tional corn. In 2005 alone, glyphosate-tolerant corn reduced the herbicide use in corn by 18.3 million pounds of active ingredient and reduced weed management costs by $238. 2 million (Sankula, 2006).

Brookes and Barfoot (2006) comp ared a typical herbicide tr eatment regime for glyphosatetolerant corn and conventional corn. They calculated the herbicide use rate and the Environmental Impact Quotient (EIQ), a measur e of impact to the environment. The EIQ integrates the various environmental impacts of a single pesticide into a single field value per hectare. The EIQ value is then multiplied by the amount of pesticide used per hectare to get the overall EIQ rating. A lower EIQ rating indi cates a lower impact on the environment.

The conventional herbicide program used a mix of herbicides, su ch as acetochlor, atrazine, primsulfuron, and dicamba. The average herbic ide use rate by growers of conventional corn was 3.74 kg active ingredient (ai)/hectare (ha) , with a field EIQ rating of 76.6/ha. The glyphosate-tolerant corn program used glyphosate plus lower rates of acetochlor and atrazine than those used on conventional crops. The va lues for the glyphosate-tolerant corn program included an average herbicide use rate of 2.59 kg ai/ha, with a field EI Q rating of 48.4/ha.

Based on these analyses, the glyphosate-toleran t corn program has a significantly better environmental profile than the c onventional herbic ide program.

19 http://www.aphis.usda.gov/b rs/aphisdocs/04_12501p.pdfMonsanto Company 09-SY-201U Page 405 of 471 N.4.3 Cotton

For a description of weed control practices in conventional cotton, pl ease see USDA petition 04-086-01p 20

. For glyphosate-tolerant cotton, ty pical weed management programs consist of a preemergence application followed by postemergence applicatio ns of glyphosate or a glyphosate only system for pre- and postemergence applications. The ove rall impact of herbicide-tolerant cotton on U.S. agriculture in 2005 alone was a reducti on in crop production costs of $39 million and pesticide use of 18 million pounds of active ingredient (Sankula, 2006).

According to Brooks and Barfoot (2006), the use of herbicide-tolerant cotton 21 (of which 98% was tolerant to glyphosate) led to reduced prod uction costs and increased profitability from
1997 to 2005 of between $21/ha an d $49/ha annually. The net gain to farm income in 2005 was $161 million. Since glyphosate-tolerant co tton was first commerci ally produced in 1997,
the farm income benefit has been $919 million. In terms of added value, the effect on farm income in 2005 was equivalent to an incr ease in production of 3% (151,000 tons).

N.4.4 Soybean

A description of herbicide use in conventio nal soybean is available in USDA petition

06-178-01P 22

. Conventional herbicide programs typically require a preemergence applica tion with one to two herbicides, followed by a postemergence applicati on with one to two herbicides. Conversely, glyphosate-tolerant soybean typica lly require only one timely appl ication of glyphosate at 0.95 lb ai/A. Consequently, in 2005, growers pl anting glyphosate-tolera nt soybean varieties reduced the overall number of herbicide applications by 39.4 million, which translated to cost savings of $134 million (Sankula, 2006). On av erage, glyphosate-tolerant soybean programs

used 1.03 lb ai/A at a cost of $21.28/A in 2005, compared to growers of conventional soybean varieties who used an additional 0.32 lb ai/A or 32% more herbicide active ingredients at an
additional cost of $18.09/A. Wh en considering herbicide costs and application costs in 2005 alone, U.S. soybean growers sa ved $1.17 billion on weed mana gement costs using glyphosate based weed-control programs (Sankula, 2006).

This reduction of herbicide use and the physi cal properties of glyphos ate have secondary environmental benefits also. Shipitalo, et al. (2008) investigated the transport loss of herbicides from agricultu re fields due to runoff. They f ound that the use of glyphosate-tolerant soybean usually, but not always, resulted in substantially less runoff than the residual herbicides that glyphosate herbic ides replaced. In contrast to the residual herbicides, the glyphosate concentrations from runoff in the watershed remained below the allowable limit for drinking water. The authors conc luded that the impact of herbic ide losses in runoff resulting from production of growing transgenic, glyp hosate-tolerant soybean should be reduced (Shipitalo, 2008).

20 http://www.aphis.usda.gov/b rs/aphisdocs/04_08601p.pdf

21 2% of the herbicide-tolerant cotto n was glufosinate-tolerant cotton.

22 http://www.aphis.usda.gov/b rs/aphisdocs/06_17801p.pdfMonsanto Company 09-SY-201U Page 406 of 471 N.4.5 Sugar beet

For a description of the weed control practices in conventional sugar beet, please see USDA petition 03-323-01p 23

. As of 2007, glyphosate-tolerant sugar beet did not have a history of widespread production, although a significant portion of the sugar beet acre s is expected to be planted with glyphosatetolerant varieties in 2009. USDA-NASS data are not yet availa ble on adoption rates, impacts on herbicide use rates, and other relevant parameters, a predictive ex ante assessment of potential impact is included in Appendix A. Coyette et al. ( 2002) predicted that introduction of glyphosate-tolerance technology in sugar beet cultiv ation in Europe would lead to a reduction in herbicide use by as much as 43%. Based on this study and data from other glyphosatetolerant crops, beneficial results in cost sa vings, pesticide reduction, and weed control are expected.

N.4.6 Bentgrass

Glyphosate-tolerant creeping bent grass is currently under review for deregulation and APHIS is conducting an environmental impact stat ement (EIS) to support its decision-making under

NEPA.

The stated rationale for deve loping glyphosate-tolerant creepi ng bentgrass event ASR368 was to benefit current agronomic practices in creep ing bentgrass seed production and its use as a principal turf on golf course tees, greens, and fairways. Control and management of annual and perennial grass, broadleaf, and sedge weed species that invade gol f courses has met with variable success using a variety of herbicides an d plant growth regulators. A discussion of the pesticides used on conventional bentgrass is available in USDA petition 03-104-01p, p. 290 24

. The most troublesome of these weeds includes annual bluegrass, rough stalk bluegrass, and bermuda grass. Considerable investments in labor, chemicals, water, and time are made to manage creeping bentgrass in golf course fairways and putting greens invaded by annual bluegrass because of bentgrass’ susceptibility to disease, heat, and drought stress. Grassy weeds occurring in creeping bentgrass seed produ ction areas can reduce th e purity of harvested seed, and can limit the land on which such seed production can profitably o ccur. Golf courses and seed producers who adopt glyphosate-tolerant creeping bentgrass in stead of conventional creeping bentgrass varieties coul d use the Roundup family of i ndustrial turf and ornamental herbicides as needed (pending U.S. Environmental Protection Ag ency label change approvals) as an effective, new, over-the-top method to control the majority of annual and perennial grasses and broadleaf weeds.

N.4.7 Alfalfa

For a description of the weed control practices in conventi onal alfalfa, please see USDA petition 04-110-01p 25

.

23 http://www.aphis.usda.gov/b rs/aphisdocs/03_32301p.pdf

24 http://www.aphis.usda.gov/b rs/aphisdocs/03_10401p.pdf

25 http://www.aphis.usda.gov/b rs/aphisdocs/04_11001p.pdfMonsanto Company 09-SY-201U Page 407 of 471 Deregulation of glyphosate-tolera nt alfalfa likely would result in significant economic and

environmental benefits from annual or se mi-annual Roundup herbicid e applications to glyphosate-tolerant alfalfa 26 . Forage quality is an important fa ctor in the price paid for hay and the presence of weeds in the hay will reduce th e quality considerably. For example, it is estimated that two applications of glyphosate at 0.75 lb a.i. /acre would provide effective season-long control of troublesome w eeds in California alfalfa fields . It is estimated that the Roundup herbicide cost would be $15/A, and a seed premium of $5/A woul d be charged. As mentioned previously, glyphosate is well rec ognized for the broad spectrum of weeds it controls. In fact, if a grower were to use a season-long herbic ide control program that provides the same spectrum and performance as glyphosate , it would require the use of trifluralin, EPTC, and imazethapyr. The level of performa nce provided by these three herbicides would be full control of 38 species, pa rtial control of eight species, and no control of two species found in California alfalfa seed production fields . This weed control program would require 4.7 lb. a.i./acre/yr. at a cost of $45/acre. Use of the glyphosate -tolerant alfalfa weed control program would provide full control of 42 species, partial contro l of 4 species, no control of one species with undetermined control of the rema ining one species (Unive rsity of California, 2001). Compared to the three herbicide program described above, the gl yphosate weed control program would not only provide broader weed control but it would reduce herbicide inputs by

3.2 lb. a.i./acre and reduce costs by $25/acre.

The use of glyphosate-tolerant crops has resulted in a drastic reduction in the amount of total herbicide applied and a change in the herbicid e active ingredient applied to U.S. cropland (Fernandez-Cornejo and Caswe ll, 2006). Deregulation of gl yphosate-tolerant alfalfa and concomitant in-crop use of glyphosate is expected to reduce the total amount of herbicide use in alfalfa production. Therefore, the cumula tive impact from deregulation of glyphosatetolerant alfalfa is expect ed to be a further redu ction in the amount of he rbicide applied to U.S. cropland.

N.4.8 Other Analyses on Herbicide Us e and Impacts from Commercial Use of Glyphosate-Tolerant Crops

Benbrook (2004) analyzed the USDA-NASS data from 1996 to 2004 and concluded that herbicide-tolerant crops have increased herbicide use by 138 million pounds with a 5%

increase in herbicide use acro ss the three major crops (corn, cotton, and soybean). Benbrook

stated that the relian ce on a single herbicide, glyphosate, as the primary method for managing

weeds on millions of acre s planted to herbicide-tolerant va rieties remains the primary factor that has led to the need to apply more herbicid es per acre to achieve the same level of weed control. These conclusions by Benbrook are not supported by the findings of Brooks and Barfoot (2006), Sankula (2006) or the USDA-ERS (2007).

USDA-ERS conducted a study entitled, “The First Decade of Genetically Engineered Crops in the United States” (Fernandez-Cornejo and Ca swell, 2006). In 2001, 2002, and 2003 USDAERS surveys, growers responded that they adop ted herbicide-tolerant crops for the following reasons: increased yields (60 – 67% of the resp onses), reduced pesticide input costs (11 – 17% of the responses), and decreased management time/ease of operation (15 – 26% of the

26 USDA Petition 04-110-01p, Section D.6., p. 282, http://www.aphis.usda.gov/brs/aphisdocs/04_11001p.pdfMonsanto Company 09-SY-201U Page 408 of 471 responses). This analysis found a cumulative re duction in herbicide use coinciding with the adoption of herbicide-tolerant crops. In addition, the report stat ed that glyphosate used on glyphosate-tolerant crops is less th an one-third as toxi c to humans and that glyphosate is not as likely to persist in the environment as the herbicides it replaces (Fernandez-Cornejo and McBride, 2002).

Based on the adoption rates of ot her glyphosate-tolerant crops, it is expected that glyphosatetolerant alfalfa would replace a si gnificant portion of conventional alfalfa varieties, especially where alfalfa is highly manage d (e.g., Western and Southwestern states). As glyphosatetolerant alfalfa is adop ted, it is expected that Roundup herb icide will replace other forms of weed control currently used in alfalfa, as discussed below.

Glyphosate-tolerant alfalfa is hi ghly tolerant to glyphosate and s hows no yield loss or loss in forage quality when treated over multiple seasons over and above maximum glyphosate application rates (Pierson and Reyes, 2006) . Recommended use ra tes are provided by Monsanto to growers in the Monsanto Technology Use Guide and Regional Technical Bulletins (See Appendix I of USDA petition 04- 110-01p). In-season maximum application of glyphosate is up to 1.5 lbs a.e. per acre at a single application, and total per-year applications is 4.5 lbs a.e./acre. These application rates we re used by Monsanto wh en conducting residue studies and for event selection pur poses. In practice, these maxi mum rates will rarely be used since the vast majority of w eeds are controlled using reco mmended rates and it would be uneconomical for growers to apply excess herbicide.

N.5 Impact on Soil Tillage Practices

Tillage is a farming practice used to prepare th e seed bed and to mechanically remove weeds from the field. No-till farming, also known as conservation tillage or zero tillage, is defined as planting and growing crops with out disturbing the soil through tillage. No-till and other conservation tillage farming practices improve soil and air quality, minimize surface runoff and soil erosion, enhance water quality, and reduce contributions to the greenhouse gases effect, particularly carbon dioxide (Ferna ndez-Cornejo and McBride, 2002; Le ep et al., 2003). No-till seeding reduces equipment use, which can result in economic benefits of reduced labor and fuel cost. In the dryland areas of the northwe st U.S., no-till seeding helps conserve soil moisture (G. Shewmaker, Extension Forage Specialist, University of Idaho, Personal Communications, 2007).

The main difference between no-till and conventional tillage seed practices is that no tillage is used to prepare the seedbed fo r seeding. When no-till seedi ng a field, a postemergence or burndown herbicide is applied prio r to or at seeding to contro l any emerged weeds in place of tillage. Herbicide-tolerant crops facilitate the adoption of no-till farming; consequently, no-till farming has been extensively adopted for co rn, cotton and soybean, for which herbicidetolerant varieties are readily available. Data av ailable for individual crops are discussed below.

N.5.1 Corn

As the adoption and commercial use of glyphosate-tolerant corn has increased, no-till corn acreage has steadily and significantly increased in parallel. In 2004 (the most recent year for which survey information is available), no-till pr actices were used on 19. 7% of the total cornMonsanto Company 09-SY-201U Page 409 of 471 acres, compared to zero no-till acres before glyphosate-tolerant co rn was commercially available in 1996. The positive im pacts from no-till production (suc h as reduced fuel use, soil erosion, runoff of pesticides and water, gl obal warming potential, greenhouse gas emissions, and improved wildlife habitat) will increase as the adoption of herbicide-tolerant crops continues to increase (Lee p et al, 2003; CTIC, 2004).

N.5.2 Cotton

The use of glyphosate-tolerant cotton has led to a significant increase in no-till production practices (Sankula, 2006). Total no-till cotton acres in 2004 were 2.4 million acres, or 18% of

the total cotton acres. This is an increase of 371% compared to the no-till acres in 1996. The

main reason for the increase in no-till cotton acreage since 1996 is the a doption of herbicidetolerant cotton, allowing over-the-top glyphosat e applications (Doane , 2002). Other reasons include enhanced grower awarene ss of the benefits of conservati on tillage practices, increase in fuel prices, access to better no-till equipment, a nd availability of better herbicides to control

weeds in no-till fields. Assu ming that the entire no-till cotton acreage in 2004 (2.4 million acres) was planted to herbicide-tolerant varieties, fuel and labor cost savings were estimated to be $48 million (Sankula, 2006).

N.5.3 Soybean

In 1995, before the introduction of glyphosate-to lerant soybean, approximately 27% of the U.S. soybean acres used no-till production. By 2004, no-till acres increase d to 36% of the total soybean acres (Sankula, 2006). A fe w states provide statistics on the adoption of no-till acres in their state. For 2007 in Indiana, no-till soybean was planted on 69% of the total soybean

acres 27 . In 2006 in Illinois, no-till farming wa s used on 51% of the soybean acres. The University of Illinois Extension Service attributed that figure to the fact that 90% of the state’s acres were planted to glyphosate-to lerant varieties, along with othe r factors, such as high fuel prices, improved equipment, highe r yields using no-till practices , and better grower awareness of the advantages to soil and water quality from no-till farming 28

.

N.5.4 Alfalfa

According to forage specialists, all alfalfa-gr owing regions currently practice some level of notill seeding (J. Caddel, Forage Specialist, Oklahoma State University, Personal Communications, 2007; D. Hancock, Forage Speci alist, University of Georgia, Personal Communications, 2007; D. Putnam, Extension Fora ge Specialist, Univer sity of California, Personal Communications, 2007; G. Shewmaker, Ex tension Forage Specialist, University of Idaho, Personal Communications, 2007; M. Sulc, Extension Forage Specialist, Ohio State University, Personal Communicatio ns, 2007; and D. Undersander, Extension Forage Specialist, University of Idaho, Personal Communications, 2007). All soil management groups (soil class

27 Summed from State of Indian a county level data from at http://www.in.gov/isda/files/So ybean_Rank_Percentage.pdf

28 University of Illinois Extension Serv ive News Release: No-till is now the "Conventional" Tillage System for Illinois Farmers; http://web.extension.uiu c.edu/state/newsdetail.cfm?NewsID=4991Monsanto Company 09-SY-201U Page 410 of 471 1 to 5) and all textures are suitable for no-till es tablishment and production provided the soils are naturally well-dr ained or tilled (Leep et al., 2003).

Few peer-reviewed publications ar e available to quantify the curr ent acreage of no- till seeded alfalfa. Therefore, personal communication with local extension forage and weed specialists was heavily relied upon to address this question. Pennsylvania is the on ly state that conducts surveys quantifying no-till alfalfa acreage. The highest levels of no-till seeded alfalfa are practiced in the North Ce ntral, East Central, an d Southern regions of th e U.S. The amount of no-till varies significantly within these regions and states. No-till seeding in the North Central region can range from 5 to 20% of the seeded acres, with Minnesota practicing the lowest

amount of no-till seedi ng (R. Becker, Weed Specialist, Un iversity of Minnesota, Personal Communications, 2007; S. Moesc hnig, Extension Weed Speci alist, South Dakota State University, Personal Communications, 2007; P. Murphy, NRCS Wisconsin, Personal Communications, 2007; D. Undersander, Extension Forage Specialist, University of Idaho, Personal Communications, 2007). Soil compaction plus cold and wet so il conditions in the spring are the primary reasons for the low adoption rate in Minnesota. The East Central region can range from 10 to 30% no-till 29 (Leep, 2003). Ohio reported the highest percentage of notill. The no-till alfalfa acreage in the state of Pennsylvania is 21.4% based on actual survey results by the Pennsylvania NASS 30

. As with the other glyphosate-to lerant crops, the ability to us e over-the-top treatments of glyphosate is expected to facilita te the further adoption of no-ti ll practices. Deregulation of glyphosate-tolerant alfalfa, when considering the data from othe r glyphosate-tole rant crops, is expected to further increase the benefits from no-till farming. These benefits include improved soil and air quality, reduced surface runoff and soil erosion, enhanced water quality, and reduced contributions to the greenhouse ga ses effect, particul arly carbon dioxide.

N.5.5 Other Crops

Limited information is available on the use of no-till farming practices in canola; however, a survey conducted in 2001 for th e Canola Council of Canada ( 2001) did find that planting of

herbicide-tolerant canola varieties resulted in an increase in the use of no-till practices. For sugar beet, more information should be avai lable after more acres are planted in 2008; however, the presence of the herbicide-tolerant trait s hould allow for adoption of no-till practices, just as experienced fo r corn, cotton, and soybean.

N.6 Conclusions

Data show the introduction of glyphosate-tole rant crops has had no impact on the overland acres of crops planted in the U.S. The glyphosat e-tolerant trait does incr ease grower flexibility in deciding what crop to plant, but has only a minor influence on the specific crop acreage planted, compared to other market forces, such as price. The planti ng of glyphosate-tolerant crops has resulted in a substan tial decrease in the overall am ount of herbicides applied to croplands. Use of glyphosate-to lerant crops has facilitated the adoption of no-till practices, which improve soil and water quality, reduce fuel use and reduce ai r pollution. Glyphosate-

29 Sulc, M., Extension Forage Specialist, Ohio State University, Personal Communications, 2007.

30 http://www.nass.usda.gov/Sta tistics_by_State/Pennsylvania/Publicat ions/Survey_Results/tillage07_feb.pdfMonsanto Company 09-SY-201U Page 411 of 471 tolerant crops increase grower flexibility regarding crop rotati on but have minimal impacts on crop rotation.

According to USDA-ERS (2006), a doption of currently deregulated glyphosate-tolerant crops

has resulted in an overall increase in farmer profitability, farmer household income, crop yield, soil conservation, and a reduction in herbicide use. Deregulatio n of other glyphosate-tolerant crops is expected to provide similar benefits to growers and the envir onment by reducing costs, reducing herbicide use, and increasing grow er planting flexibility in crop choice.Monsanto Company 09-SY-201U Page 412 of 471 Appendix N References

Ash, M., J. Livezey, and E. Dohlman. 2006. Soybean backgrounder. U.S. Department of Agriculture-Economic Research Service, Washington, D.C.

Benbrook, C. 2004. Genetically engineered crops and pesticide use in the United States: The first nine years. BioTech InfoNet T echnical Paper No. 7. www.biotechinfo.net/Full_version_first_nine. pdf [Accessed July 2, 2009].

Brookes, G and P.Barfoot. 2006. Global impact of biotech crops: socio-economic and environmental effect in the first ten years of commercial use. AgBioForum. 9:139-151.

CCC. 2001. An agronomic and economic assessmen t of transgenic canol a. Canola Council of Canada. http://www.canola-council.org/gm o_toc.aspx [Accessed July 2, 2009].

Coyette, G., F. Tencalla, I. Br ants, Y. Fichet, and D. Rouchou ze. 2002. Effect of introducing glyphosate-tolerant sugar beet on pesticide usage in Europe. Pesticide Outlook 13:219-224.

CTIC. 2004. Crop residue management. Conserva tion Technology Information Center, West Lafayette, Indiana.

Doane Marketing Research 2002. Conservation tillage study prepared for the National Cotton Foundation. http://www.cotton.org/tech/biotech/c ontill-study.cfm [Accessed July 2, 2009].

Fernandez-Cornejo, J. and W.D. McBrid e. 2002. Adoption of bioengineered crops. Agricultural Economic Report AE R810, U.S. Department of Ag riculture, Washington, D.C.

Fernandez-Cornejo, J. and M. Caswell. 2006. Th e first decade of genetic ally engineered crops in the United States. Economic Information Bulleting E1B-11, U.S. Department of Agriculture, Agricultural Economic Re port, Washington, D.C.

Leep, R., D. Undersander, P. Peterson, D-H Mi n, T. Harrigan, and J. Grigar. 2003. Steps to successful no-till establishment of forages. Mich igan State University, University of Wisconsin, University of Minnesota, and Natural Resources Conservation Service, Extension Bulletin E2880.

Monsanto. 2007. Monsanto biotechnology trait ac reage: Fiscal years 1996 to 2007. Monsanto Company, St. Louis, Missouri.

Pierson, P. and C. Reyes. 2006. Roundup Ready alfa lfa: Summary of third- party data for yield, forage quality and crop safety. National Meeti ng of the North American Alfalfa Improvement

Conference.

Sankula, S. 2006. Quantification of the impact s on US agriculture of biotechnology-derived crops planted in 2005. National Center for Food and Agriculture Policy, Washington, D.C.

Shipitalo, M.J., R.W. Malone, and L.B. Owens. 2008. Impact of glyphosate-tolerant soybean and glufosinate-tolerant corn production on he rbicide losses in surface runoff. Journal of Environmental Quality 37:401-408.Monsanto Company 09-SY-201U Page 413 of 471 UC. 2001. Alfalfa: Susceptibility of weeds to herb icide control. University of California Pest Management Guidelines. http://www.ip m.ucdavis.edu/PMG/r1700411.html#WINTER [Accessed July 2, 2009].

USDA-ERS. 2007. Adoption of genetic ally engineered crops in th e U.S. U.S. Department of Agriculture, Washington, D.C

USDA-ERS. 2006. Soybean production costs and re turns per planted cr op acre, by region, excluding government payments for 2006. U.S. De partment of Agriculture-Economic Research Service. (http://www.ers.usda.gov/ data/costsandreturns/Soy_all.xl s) [Accessed June 26, 2009].

USDA-NASS. 2006. Crop production 2005 summar y. U.S. Department of Agriculture National Agricultural Statistics Service.

USDA-NASS. 2007. Crop production historical track records. http://usda.mannlib.cornell.edu/usda/cur rent/htrcp/ [Accessed July 16, 2009]

Vesterby, M. and K.S. Krupa. 1997. Major uses of land in the Un ited States. Statistical Bulletin 973. U.S. Department of Agri culture, Washington, D.CMonsanto Company 09-SY-201U Page 414 of 471 Appendix O. Potential Impact of Glyphosa te on Human Health and the Environment

O.1 Overview

Glyphosate is a herbicide approved for use (regi stered) by the U.S. Environmental Protection Agency (EPA or the agency) for the control of weeds that would interfere with the growth of many food and non-food crops, in cluding biotechnology-de rived crops, as well as for control of weeds growing in non-crop areas. In 2001, EPA identified glyphosate as the most widely used conventional agricultural pe sticide in the U.S. (Kiely et al., 2004). Glyphosate has been registered, and food and f eed tolerances have been established in the U.S. for its residues, since 1979. Glyphosate has also successfully complete d the reregistration pro cess, as required for the continued registration of all pesticides originally registered before 1984.

Glyphosate has a complete and comprehensive regul atory data base (toxicity, environmental fate, and ecological toxicity) th at has been evaluated by EPA to support all currently approved uses including glyphosate-tolerant al falfa. EPA has stated that it has a high level of confidence in the quality of the existing studies and the re liability of the toxicity endpoints that are the basis for risk assessment (EPA, 200 6a,b,c). In establishing food and feed tolerances to support the use of glyphosate on animal feed and forage crops (the group tolerance that supports the use of glyphosate in conventional and glyphosate- tolerant alfalfa), EP A noted that it had conducted “a complete and thorough review of the available data for glyphosate,” and determined that “glyphosate will not pose unreas onable risks or adverse effects to humans or the environment” (EPA, 2002 31 ).

The following discussion provides an overview of the regulatory and risk assessment processes applicable to glyphosate and all other agri cultural use pesticides. Glyphosate has been approved by the EPA for a large number of food and feed uses, including uses associated with glyphosate-tolerant crops. Over 180 food and fe ed tolerances (40 CFR § 180.364) have been established for glyphosate in suppo rt of these uses. A complete listing of all U.S. glyphosate tolerances is provided in Attachment 1.

O.1.1 Pesticide Registration and Tolerance Setting

The Federal Insecticide, Fungicide, and Rodentic ide Act (FIFRA) requires that before sale or distribution of a pesticide in the U.S., a person or company must obtain a registration, or license, from EPA. Before regi stering a new pesticide or a new use for a previously registered pesticide, EPA must first ensure that the pestic ide, when used according to its label directions,

will not cause unreasonable adverse effects on the environment. In order to address this standard, EPA must evaluate potential risks to humans and the environment, and may require applicants to submit more than 100 different scie ntific studies and test s conducted according to EPA guidelines. According to EPA, glyphosate is one of more than 1055 active ingredients currently registered as pestic ides, which are formulated into many thousands of pesticide products that are available in the marketplace.

The process of registering a pesticide is a sc ientific, legal, and administrative procedure through which EPA examines the ingredients of the pesticide; the particul ar site or crop on

31 Prior to 2002, separate alfalfa forage and alfalf a hay tolerances were es tablished for glyphosate.Monsanto Company 09-SY-201U Page 415 of 471 which it is to be used; the amount, frequency, method and timing of application, and other conditions of its use; and storag e and disposal practices. In ev aluating a pesticide registration application, EPA assesses a wide variety of potential human hea lth and environmental effects associated with use of the product.

The data required by EPA are used to evaluate whether a pesticide has the potential to cause adverse effects on humans, wildlife, fish, and pl ants (including endange red species and NTOs, organisms that the pesticide is not intended to ac t against). The registration applicant must also supply data addressing the pesticide’s poten tial impact on surface water or ground water (which might result from leachi ng or runoff, for example). Pote ntial human health and safety risks range from short-term toxicity to l ong-term effects such as cancer and reproductive system disorders.

EPA also must approve the language that appe ars on each pesticide label. A pesticide product can only be used legally according to the dire ctions for use on the labeling accompanying it at the time of sale. Following these directions carefully and precisely is necessary to ensure safe

use.

A pesticide’s registration is not the only opportunity EPA has to eval uate that product’s safety. For example, EPA is currently completing a one -time program to revi ew older pesticides (those initially registered be fore November 1984) under FIFRA to ensure that they meet current scientific and regulatory standards. This process, calle d reregistration, considers the human health and ecological effects of pesticides and results in actions to reduce risks that are

of concern. EPA concluded it s reregistration evalua tion of glyphosate in 1993. At that time, the EPA produced a 291-page Reregistratio n Eligibility Decision document (RED) on glyphosate, setting forth the data on which it ma de a decision to reregi ster all then-existing uses of the pesticide, based on the pesticid e having met the no unreasonable adverse effects

standard found in FIFRA.

Where pesticides may be used on food or feed crops, EPA also sets tolerances (maximum pesticide residue levels) for the am ount of the pesticide that can legally remain in or on foods. EPA undertakes this analysis under the authorit y of the Federal Food, Drug, and Cosmetic Act (FFDCA). Under the FFDCA, EPA must find that such tolerances will be safe, meaning that there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue. This finding must be made an d the appropriate tolera nce established before a pesticide can be registered fo r use on the particular food or f eed crop in question. Several

factors must be addressed before a tole rance can be established, including:

• the aggregate, non-occupational exposure from the pesticide (exposure through diet, from using pesticides in and around the home, and from drinking water);

• the cumulative effects from exposure to diffe rent pesticides that produce similar effects in the human body;

• whether there is increased susceptibility to infants and children, or other sensitive subpopulations, from exposure to the pesticide; and

• whether the pesticide produces an effect in humans similar to an effect produced by a naturally occurring estrogen or produces other endocri ne-disruption effects.Monsanto Company 09-SY-201U Page 416 of 471 O.1.2 Pesticide Risk Assessment

The process EPA uses for evaluating the health impacts of a pesticide, under either FIFRA or the FFDCA, is called risk assessment. EPA us es the National Research Council’s four-step process for human health risk assessment, which involves hazar d identification, dose-response assessment, exposure assessment and risk characte rization. Each of th ese steps is discussed

below:

The first step in the risk assessment process is to identify potential health effects, or hazards that may occur from different types of pesticid e exposure. EPA considers the full spectrum of a pesticide’s potential health e ffects. Hazards are identified th rough a battery of studies that examine the potential toxicity of the pesticide in various tests including, where appropriate, tests with laboratory animals.

Generally, for human health risk assessments , many toxicity studies are conducted, based on EPA guidelines, by pesticide companies in independent laboratories following the Good Laboratory Practice (GLP) standard s, and evaluated for acceptability by EPA scientists. EPA evaluates pesticides for a wide range of effects, from eye a nd skin irritation to cancer and birth defects. EPA may also consult the public liter ature or other sources of information on any aspect of the chemical.

The next step of the risk assessment consider s the levels at which the pesticide produces adverse effects. Dose-response assessment i nvolves considering the dose levels at which adverse effects were observed in test animals, an d using these dose levels to calculate an equal

dose in humans.

Step three of the process invo lves an exposure assessment. People can be exposed to pesticides in three ways:

1. Inhaling pesticides (i nhalation exposure),

2. Absorbing pesticides through the skin (dermal exposure), and

3. Ingesting pesticides (oral exposure).

Depending on the situation, pestic ides could enter the body by any one or all of these routes. Typical sources of pesticide exposure include agri cultural (food); home and personal use pesticides; pesticides applied to lands that make their way into the drinking water; or occupational exposure for agricultural wo rkers or pesticide applicators.

Risk characterization is the final step in assessin g human health risks from pesticides. It is the process of combining the hazard, dose-response and exposure assessments to describe the overall risk from the use of a pe sticide. It explains the assump tions used in assessing exposure as well as the uncertainties that are built into the dose-response assessment. The strength of the overall database is considered, and broad conclusions are made. EP A’s role is to evaluate both toxicity and exposure and to de termine the risk associated wi th use of the pesticide.

The risk to human health from pesticide exposur e depends on both the toxicity of the pesticide

and the likelihood of people coming into contact with it (exposure) . At least some exposure and some toxicity are required to result in a risk. For example, if the pesticide is found to have a high level of toxicity, but people are not exposed to the pes ticide, there is no risk. Likewise,Monsanto Company 09-SY-201U Page 417 of 471 if there is ample exposure but the pesticide is nontoxic, there is no risk. However, usually when pesticides are used , there is some toxicity and exposure, which results in a potential risk.

EPA recognizes that effects vary between animals of different species and from person to person. To account for this variability, a 100-fold uncertainty factor is built into the risk assessment. This uncertainty factor creates an additional margin of safety for protecting people who may be exposed to the pest icides. FQPA requires EPA to use an extra 10-fold safety factor, if necessary, to prot ect infants and children from effects of the pesticide.

Once EPA completes the risk assessment process for a pesticide, the Agency uses this information to determine if (w hen used according to label dire ctions), there is a reasonable certainty that the pesticide will not harm a person’s health.

Using the conclusions of a risk assessment, EPA can then make a more informed decision regarding whether to approve a pesticide chemical or use, as proposed, or whether additional protective measures are necessa ry to limit occupational or non-occupational exposure to a pesticide. For example, EP A may prohibit a pesticide from being used on certain crops because consuming that commodity treated with the pesticide may result in an unacceptable risk to consumers. Another example of prot ective measures is requiring workers to wear personal protective equipment (PPE) such as a resp irator or chemical resistant gloves, or not allowing workers to enter treated crop fields until a specific period of time has elapsed.

If, after considering all appropriate risk reduc tion measures, the pesticide still does not meet EPA’s safety standard, the Agency will not allow the proposed chemical or use. Regardless of the specific measures enforced, EP A’s primary goal is to ensure that legal uses of the pesticide are protective of human health, especially the health of chil dren, and the environment.

O.2 Potential Impact of Glyphosate on Human Health

O.2.1 Glyphosate Safety Evaluations

Glyphosate presently has 186 establ ished food and feed tolerances in the U.S (see Attachment 1). Each time EPA reviews an application to add a new food or feed use to the glyphosate label the Agency is required by FFDCA to cond uct an aggregate risk assessment, considering all non-occupational sources of human exposure to the pesticide, and find that aggregate exposure to the pesticide will be safe as de fined by the statute and regulations. Issues associated with potential occupational exposure for each new use are considered under

FIFRA’s unreasonable risk standard.

Over the course of these numerous reviews, the toxicology of glyphosat e has been extensively studied. Comprehensive toxicol ogical studies in an imals have demonstrated that glyphosate does not cause cancer, birth defect s, mutagenic effects, nervous system effects or reproductive problems 32 (EPA, 1993; WHO/FAO, 2004). In fact, af ter a thorough review of all available

32 European Commission (EC ). 2002. Report for the Active Substance Glyphosate, Direct ive 6511/VI/99, January 21. http://europa.eu.int/comm/food/fs/ ph_ps/pro/eva/existing/list1_en.htm

.Monsanto Company 09-SY-201U Page 418 of 471 toxicology data, the EPA conclude d that glyphosate should be cl assified in Group E - Evidence of Non-carcinogenicity in Humans, the most favorable category po ssible (EPA, 1993).

Despite this extensive sa fety data, glyphosate safety is reviewed with every new use for which registration is sought, in cluding, where necessary, uses asso ciated with glyphosate-tolerant crops developed through biotechnolo gy. As discussed above, prior to the approval of any new use of an existing registered pesticide, EPA mu st consider the potential human health effects from the aggregate (total combined) human expos ure to that pe sticide, combin ing the potential exposure from the proposed new use with all other existing exposures to the pesticide. Dietary exposure is considered, which addresses pesticid e residues that may remain on food from crops on which the pesticide is applied (pre- or posteme rgence), as well as any residue that could be found in drinking water as a result of pesticide use. Non-dietar y exposure is also included in this assessment, which includes exposure to th e pesticide through reside ntial use, such as on lawns or in flower beds, as well as exposure in a recreational context, such as from a golf course or sports field. Based on these data, EPA must be able to make a determination of reasonable certainty of no harm to human health as required by the FFDCA.

EPA does not conduct an acute dietary risk asse ssment for glyphosate because no acute effects

have ever been identifie d in the toxicological studies conduct ed for glyphosate. Accordingly, EPA does not expect glyphosate to pose an acute risk (EPA, 2006d). EPA does conduct a chronic dietary (food and water) risk assessmen t for glyphosate based on a theoretical worst case exposure estimate. For food, this estimate assumes that glyphosate is used on 100 percent of all the crops on which the pesticide is currentl y approved for use. It further assumes that the resulting pesticide residues found on all harves ted food crops are at the level of the legally established tolerance (i.e., the maximum allowabl e pesticide residue level). For water, EPA assumes that glyphosate is used to control weeds in water bodies by direct application to the water at the maximum applicati on rate, without taking into acc ount degradation in the water body (EPA, 2006b,c).

Applying this unrealistic, theo retical maximum exposure estimate, EPA determines how much

of the established Reference Do se (RfD) would be utilized by all currently approved product

uses. The RfD is an estimate of the amoun t of daily pesticide exposure to the human population that can occur over a lifetime with a reasonable certainty of no harm to human

health. 33 For glyphosate, the RfD is 1.75 mg pe r kg body weight per da y (mg/kg/day) (EPA, 2002). Provided that the utiliz ation of the RfD from food and dr inking water does not exceed the EPA level of concern of 100 percent 34 , 35 , EPA will then conduct an aggregate risk assessment (chronic and short/in termediate-term) to consider additional exposures from nondietary exposure routes (residential and recreational).

If the aggregate risk assessment shows that utilization of the RfD does not exceed the EPA level of concern, then EPA will conclude that the new use does not pose an unreasonable risk

to human health. EPA will then establish or revise, as needed, any food or animal feed crop

33 RfD is the current terminology used by EPA; however earlier EPA risk assessment terminology used the term Allowable Daily Intake (ADI). RfD and ADI are synonymous.

34 EPA has reviewed state monitoring data on the occurrence of glyphosate in public drinking water systems and found that these data “reinforce the Agency’s conclusi on that aggregate exposure to glyphosat e via all exposure rout es, including drinking water, will not exceed the Agency’s level of concern” (U.S. EPA, 2002).

35 For glyphosate, the EPA’s level of concern for ch ronic dietary exposure is 100 (U.S. EPA , 2006b).Monsanto Company 09-SY-201U Page 419 of 471 tolerances to allow for the pr esence of glyphosate residue on that crop. EPA publishes these

new tolerances in the Federal Register, along with a summary of th e risk assessment and approves pesticide labeling for the new use. In issuing the final tolerance rule, EPA considers and discusses any comments received in respons e to the original no tice regarding EPA’s

intention to establish tolerances that was published in the Federal Register.

Despite the large number of approved food and feed uses of glyphosate, including uses associated with glyphosate-tolerant crops, a large margin of safety exists for glyphosate. While use of glyphosate has increased in the decade since the in troduction of glyphosate-tolerant crops, the associated risk to human health as a result of the increased human exposure to glyphosate remains low, due to the low mammalia n toxicity of glyphosat e and the relatively low dietary exposure associated with the herbicide’s approved uses.

Prior to the first approv al of a glyphosate-tolerant crop (so ybean) in 1996, theoretical dietary exposure for all registered conventional uses of glyphosate utilized approximately 2.9% of the glyphosate RfD for the most sensitive subpopulati on of non-nursing infants less than one year old (EPA, 1993). Incremental theoretical diet ary exposure from the additional uses of glyphosate in glyphosate-tolerant soybean, cotton, corn, canola, alfalfa, sugar beet and creeping bentgrass combined wi th all other conventional cr op uses of glyphosate approved between 1996 and 2006, account for only an add itional 1% utilization of the glyphosate RfD for the subpopulation of infant s less than one year old (EPA , 2004d). The total combined chronic aggregate exposur e (dietary and non-dietary) from al l uses of glyphosate utilizes well below 100% of the glyphosate RfD, only 9% fo r the most sensitive subpopulation of nonnursing infants less than one y ear old (EPA, 2006b). The total combined short/intermediate term aggregate exposure (dietary and non-dietary) for all current regist ered uses of glyphosate utilizes only 11% of the glyphosate RfD for th e most sensitive subpopulation of non-nursing infants less than one year old (EPA, 2006b). The utilization of the glyphosate RfD, which is well below 100 percent, has allowed EPA to con tinue to make the conclusion of reasonable certainty of no harm to human health for each glyphosate use, including glyphosate-tolerant alfalfa and other new gly phosate-tolerant crop uses.

These figures are supported by the data provided in the tables be low. Table O-1 summarizes the established food and feed tolerances suppor ting the use of glyphosate in the conventional crops of alfalfa, cotton, sugar beet and soybean prior to the first glyph osate-tolerant crop in 1996. A summary of the regula tory approvals, including new or modified food and feed tolerances, and associated dietary exposure asse ssments for approved gly phosate-tolerant crops is provided in Table O-2. Table O- 3 summarizes the most recent chronic and short/intermediate-term aggregate ri sk assessments for glyphosate.Monsanto Company 09-SY-201U Page 420 of 471 Table O-1. Established Glyphosate Toleran ces Prior to Glyphosate-tolerant Crops
(1993)

Crop Established Food/Feed Tolerances Publication % of Reference Dose (RfD)

Soybean • Seed – 20 ppm

• forage & hay – 15 ppm

• hulls – 100 ppm Glyphosate Reregistration Eligibility Decision

Document

September 1993

(EPA, 1993) General Population - 1.2

Non-nursing infants <1 year old - 2.9

Alfalfa 200 ppm

Cotton forage, hay, & seed – 15 ppm

Sugar beet Roots – 0.2 ppmMonsanto Company 09-SY-201U Page 421 of 471 Table O-2 (continued). Summary of EPA Approvals for Glyphosate Use in Glyphosatetolerant Crops

Commercial

Introduction

Year Required Changes in Food/Feed

Tolerances Federal Register

Publication Establishing New or

Modified Tolerance % of Reference Dose (RfD)

Dietary Exposure Only

(Food + Water)

Roundup Ready soybean 1996 • Increase soybean forage to 100

ppm.

• Increase soybean hay to 200

ppm.

• Establish new tolerance for aspirated grain fractions at 50

ppm. 61 FR 15192

Petition No. 4F4369

Apr. 1996

(EPA, 1996b) General Population – 1

Non-nursing infants- 2.5

Roundup Ready

cotton 1997 Establish new tolerance for gin byproduct at 100 ppm. 61 FR 7729

Petition No. 5F4493

Feb. 1996

(EPA, 1996a) General Population - 1

Non-nursing infants - 2.4

Roundup Ready

corn 1998 Establish new tolerance for corn forage at 1 ppm. 62 FR 17723

Petition No. 5F4555

Apr. 1997

(EPA, 1997) General Population - 1

Non-nursing infants < 1 year old

- 3

Roundup Ready

canola 1999 Establish new tolerances for

canola.

• seed at 10 ppm

• meal at 15 ppm 64 FR 18360

Petition No. 2E4118

Apr. 1999

(EPA, 1999) General Population - 1.5

Non-nursing infants <1 year old

- 3.3

Roundup Ready sugar beet 2008 Establish new tolerances for sugar

beet.

• roots at 10 ppm

• tops at 10 ppm

• pulp (dried) at 25 ppm

Roundup Ready

corn 2 2004 Increased tolerance for corn forage to 6 ppm. 68 FR 36472

Jun. 2003

(EPA, 2003) Change in forage tolerance did not affect estimated dietary exposure from animal products; therefore no dietary risk

assessment was conducted.

Roundup Ready

Flex cotton 2006 • Increase tolerance for gin byproduct to 175 ppm.

• Increase tolerance for cottonseed to 35 ppm. 69 FR 65081

Petition No. 3F6570

Nov. 2004

(EPA, 2004d) General Population - 2.2

All infants < 1 year old - -3.9

Roundup Ready

alfalfa 2006 Establish new tolerances for alfalfa seed at 0.5 ppm.

Existing tolerances for alfalfa forage (400 ppm) and hay (200 ppm) were sufficient to cover new in-crop uses. 70 FR 7861

Petition No. 2F6487

Feb. 2005

(EPA, 2005) 1 Dietary exposure insignificant,

did not conduct new risk

assessment. Deferred to

assessment conducted for flex cotton as published in 69 FR

65081.

1 U.S. Environmental Protection Agency. 2005. Final Rule . Federal Register Vol. 70, No. 31: 7861. Feb. 2005.Monsanto Company 09-SY-201U Page 422 of 471 Table O-3. Aggregate Exposu re Assessment for Glyphosate

Population Subgroup Acute Aggregate 2 RfD (mg/kg/day) 2 Chronic Aggregate 1,2 Short/Intermediate Term Aggregate ,2

Exposur

e (mg/kg/

day) % RfD Exposure (mg/kg/day) % RfD

General U.S. population

Not applicable 1.75 0.041 2 - -

All infants (<1 year) 0.127 7 0.157 9

Non-nursing infants (<1 year) 0.158 9 0.188 11

Children 1-2

years 0.095 5 0.125 7

Children 3-5

years 0.088 5 0.118 7

Children 6-12

years 0.059 3 0.089 5

Youth 13-19

years 0.037 2 - -

Adults 20-49

years 0.033 2 0.063 4

Adults 50+ years 0.028 2 - -

Females 13-49

years 0.031 2 - -

1 Chronic aggregate exposure is the same as chr onic dietary exposure becau se chronic non-dietary

exposure is not expected based upon the curre nt registered non-crop uses of glyphosate.

2 EPA OPPTS. Glyphosate Human H ealth Risk Assessment for Proposed Uses on Safflower and

Sunflower. Petition No. 4E6878. Sept. 5, 2006.

O.2.2 Glyphosate Safety Evaluation fo r Applicator and Bystander Exposure

Another potential impact of th e use of glyphosate on human hea lth that EPA considers in its human health analysis is applicator a nd bystander exposure resu lting from increased glyphosate use. Based on the toxicity of glyphos ate and its registered uses, including use on glyphosate-tolerant crops, EP A has concluded that occupational exposures (short-term dermal and inhalation) to gl yphosate are not of concern beca use no short-term dermal or inhalation toxicity endpoint s have been identified for glyphosate (EPA, 2006b; EPA, 2006c).

Additional evidence to support the EPA conclusion can be found in the Farm Family Exposure Study (Acquavella et al., 2004), a bi omonitoring study of pe sticide applicators conducted by independent investigators. This biomonitoring study determined that the highest estimated bodily adsorption of glyph osate as the result of routine labeled applications of registered glyphosate-based ag ricultural herbicides to crops, including glyphosate-tolerant crops, was approximately 400 times lower than the RfD established for glyphosate. Furthermore, investigators determined that 40% of applicators did not have detectable exposure on th e day of application, and 54 % of the applicators had anMonsanto Company 09-SY-201U Page 423 of 471 estimated bodily adsorption of glyphosate more than 1000 times lower than the RfD (Acquavella et al., 2004).

The biomonitoring study also f ound little evidence of detectab le exposure to individuals

on the farm who were not activ ely involved in or located in the immediate vicinity of labeled applications of glyphosate-based agricu ltural herbicides to crops. Considering the similarity of the use pattern and appli cation rates of the glyphos ate products in this study compared to those regist ered for use on glyphosate-tole rant alfalfa and glyphosatetolerant crops in general, bystander exposur e attributed to the use of glyphosate on glyphosate-tolerant crops is e xpected to be negligible.

O.3 Potential Impact of Glyphosate on the Environment

Potential environmental effects are carefully co nsidered as a part of the FIFRA pesticide registration process. Prior to the approval of a new pestic ide or a new use (including a change in pesticide applicati on rates and/or timing) and befo re reregistering an existing pesticide, EPA must consider the potenti al for environmental effects and make a determination that no unreasonable adverse effects to the environment will be caused by the new pesticide, new use or continued use.

To make this determination, EPA requires a comprehensive set of environmental fate and ecotoxicology data on the pest icide’s active ingredient (40 CFR Part 158). EPA uses these data to assess the pesticide’s potential environmental risk (hazard x exposure). The required data include both short and long-te rm hazard data on representative organisms that are used to predict hazards to terrestri al animals (birds, nontarget insects, and small mammals), aquatic animals (freshwater fish and invertebrates, estuarine and marine organisms), and nontarget plants (terrestrial and aquatic).

EPA reevaluated the environmental safety of glyphosate in 1993 as part of the FIFRArequired reregistration of all pesticides. At the end of this evaluation, EPA concluded that all registered uses of glyphosate were e ligible for reregistration, including terrestrial (i.e., land-based) applications up to 7.5 pounds glyphos ate acid equivalents (a.e.) per

acre.

Since the reregistration evaluation in 1993, EPA has reviewed and approved a significant number of new glyphosate uses : conventional crops such as legume vegetables and sunflower/safflower seed, glyphosate-tolerant crops such as corn, cotton, canola and soybean, and non-crop areas. In each case, EP A concluded that the new use, including any incremental environmental exposure to gl yphosate caused by that new use, did not pose an unreasonable risk to the environmen t, and approved pesticide labeling for the

new use.

The studies and data collected by Monsanto , both for the initial EPA registration and reregistration of glyphosate, as well as data developed by in dependent academics, present a well-established safety profile for glyphosat e. The following sections provide greater detail regarding some of the key findings from these studies.Monsanto Company 09-SY-201U Page 424 of 471 O.3.1 Persistence of Glyphosate in the Soil

Persistence of agricultural ch emicals in the soil is widely regarded as an undesirable

environmental characteristic. Glyphosate has been shown to ra pidly dissipate from most agricultural ecosystems across a wide range of soil and climatic conditions, with a median soil half-life (the time it takes for half of the glyphosat e to dissipate in the soil) of 13 days (Giesy et al., 2000). The potential for glyphosate to accumulate in soil following repeated applications has been studied both in the laboratory and the field.

A laboratory study was conducted on two soil samp les, with each sample receiving up to three sequential applicat ions of 5 pounds glyphosat e a.e. per acre over a 6-week period, at

two-week intervals. The concentration of glyphosate in soil 24 weeks following application had declined to 1-5% of the concentration i mmediately after application, regardless of whether it was the firs t, second or third application.

Glyphosate degradation in the soil following mu ltiple glyphosate applications was also

shown under field conditions. Soil was colle cted from pesticide efficacy and tolerance trials in orchards and vineyards that receiv ed repeated applicatio ns of glyphosate over a one- to six-year period, at cumulative rate s of 6 to 120 pounds glyphosate a.e. per acre. These soil samples did not show any accumula tion of glyphosate residues, even at the exaggerated rate of three seque ntial applications of eight pounds glypho sate a.e. per acre
within a three-month in terval for five out of six sequen tial years. Glyphosate degradation continued after multiple applications, and less than 10 percent of the total applied glyphosate remained in the soil one year af ter the last glyphosat e application.

A typical agronomic (annual) use pattern for glyphosate on glyphosate- tolerant crops is a preemergence burn down application of 1.5 pounds glyphosate acid per acre followed by one to two postemergence applications rangi ng from 0.77 to of 1.5 pounds glyphosate a.e. per acre, and where sequential applicatio n intervals are longer than two to four

weeks. The maximum labeled rates and t ypical use patterns ofglyphosate on glyphosatetolerant crops are well within the rates and fr equencies used in the studies above. As a result, glyphosate is not expected to accumulate in soil as a result of labeled uses in glyphosate-tolerant crop.

O.3.2 Persistence of Surfactant in the Soil

Pesticide products approved for application to emerged weed s normally are applied with surfactants. Glyphosate products are formul ated with surfactan ts to increase the permeability of the cuticle wax of the weed foliage to increase the foliar uptake of glyphosate. In other words, the surfactan t acts to break down the plant’s natural protective wax coating, allo wing the plant to better absorb the glyphosate, thereby improving the efficacy of the herbicide.

The predominant type of surfact ant used in formulated gly phosate products worldwide is polyethoxylated alkyl amine (POE A). When degradation of POEA was investigated in three types of soil (silt loam, silty clay loam, and sandy loam), microbial degradation was determined to be the primary degradation route, with minimal degradation occurring under sterile conditions. Approxi mately 25-30% of applied 14 C-POEA was mineralized to

14 CO 2 within seven weeks. The estimated degr adation half-life for parent POEA was lessMonsanto Company 09-SY-201U Page 425 of 471 than one week and possibly as short as one to two days. Because limited data are available for POEA dissipation, a conservative estimate of ha lf-life values for POEA in soil would be 7-14 days 36 (Giesy et al., 2000). Glyphosat e and the POEA surfactant have similar soil dissipation rates and the same prim ary route of dissipation, i.e., microbial degradation. Therefore, it is reasonable to assume that th e POEA surfactant will behave similarly to glyphosate in field soil, and an increase in residual soil concentrations (accumulation) of the POEA surfactant is not an ticipated as a result of increased use of glyphosate associated with the planting of glyphosate-tolerant crops in general.

O.3.3 Surface Water and Groundwater

Glyphosate binds strongly to soil and has a low potential to move offsite to surface water or leach to groundwater (EPA, 1993). The EPA has estimated glyphosate levels that could occur in surface water based on pres ently approved use patterns. Relying on toxicological data from acute and chronic tests on fish and other aquatic organisms, EPA has determined that “the potential for e nvironmental effects of glyphosate in surface water is minimal” (EPA, 2002).

O.3.4 Wildlife

1. Animals: As a part of the reregistration ev aluation under FIFRA, EPA conducted an ecological assessment for glyphosate. This assessment compared the results from toxicity tests with glyphosate conducted with various plant and animal species to a

conservative estimate of the c oncentration of glyphosate to which an organism might be exposed in the environment. This estimate, called the Es timated Exposure Concentration (EEC), is a point estimate for exposur e that does not take into account normal environmental dilution or dissipation, or the frequency of exposure to the pesticide by wildlife. In the Reregist ration Eligibility D ecision (RED) for glyphosate (EPA, 1993), the exposure estimates were determined assu ming an application rate of 5.0625 lb a.e.,

which exceeds the maximum labeled use rate for agricultural purposes. When the EECs were calculated for aquatic plants and animals, the direct application of this rate to water was assumed. Based on this assessment, EP A concluded that effect s to birds, mammals,

fish and invertebrates are minimal based on available data (EPA, 1993).

Glyphosate is practically nontoxi c to honey bees (which are used to assess effects on nontarget insects in general) and practically nontoxic to slightly toxic to birds, freshwater fish, marine and estuarine species, aqua tic invertebrates and mammals (EPA, 1993). Glyphosate has a low octanol-water coefficient, indicating th at it has a tendency to remain in the water phase rather than move from the water phase into fatty substances; therefore, it is not expected to accumulate in fish or other animal tissues.

36 Marvel, J.T., Brightwell, B.B. and Suba, L.A. 1974. G3780A Surfactant: Biodegradation, plant uptake and

14 C Distribution. Monsanto Company Report No. 321Monsanto Company 09-SY-201U Page 426 of 471 The glyphosate end-use products used in agricult ure contain a surfactan t to facilitate the uptake of glyphosate into the plant (Ash ton and Crafts, 1981). Depending on the surfactant used, the to xicity of the end-use product ma y range from practically nontoxic to moderately toxic to fish and aquatic i nvertebrates (EPA, 1993). For this reason, the 1993 Glyphosate RED stated that some form ulated end-use produ cts of glyphosate needed to be labeled as “Toxic to fish” if they were labeled for direct application to water

bodies. Due to the associated hazard to fish and other aquatic or ganisms, glyphosate enduse products that are labeled for applications to water bodies generally do not contain

surfactant.

2. Plants: Glyphosate is a non-selec tive herbicide with activity on essentially all annual and perennial plants. As such, exposure to glyphosate could put aq uatic and terrestrial nontarget plants as well as threatened a nd endangered plants at risk (EPA, 1993). Nontarget plants may potentially be at risk from applications of glyphosate as a result of spray drift. As discussed earlier, glyphosat e binds tightly to so il and does not move offsite. Moreover, glyphosate is not taken up from agricultural soils by plants. Therefore risks to nontarget plants are only attributed to the spray drift of the pesticide. Pesticide labels include specific risk management me asures to manage sp ray drift, including mandatory requirements for aerial applications.

During the reregistration process in 1993, additi onal data on terrestri al nontarget plants were requested by the EPA. Th ese additional data have been utilized in conjunction with an exposure assessment to further understa nd the potential risk to threatened and endangered plants from the use of glyphosate 37 (Mortensen et al., 2008 ).

O.3.5 Endangered and Threatened Species

The EPA Endangered Species Pr otection Program web site, http://www.epa.gov/espp/

, describes the EPA assessment process for endange red species. The e ssential elements of that process, generally taken from the web site, are summarized below.

The Endangered Species Act (ESA ) was intended to protect and promote the recovery of animals and plants that are in danger of becoming extinct. All federal agencies are required under the ESA to ensure that their regulatory actio ns, including EPA’s registration of pesticides in the U.S., are not likely to jeopardize the continued existence of threatened or endangered species (“list ed” species) or destroy or adversely modify

their critical habitat.

EPA's Endangered Species Protection Program (ESPP) helps promote the recovery of listed species. The ESPP is a program desi gned to determine whether pesticide use in a certain geographic area may a ffect any listed species.

When registering a pesticide or reassessing th e potential ecological risks from use of a currently registered pesticide, EPA evaluate s extensive toxicity and ecological effects

37 Mortensen, SR, Carr, KH, and Honegger, JL. 2008. Tier I Endangered Species Assessment for Agricultural Uses of Glyphosate and Glyphos ate-Containing Herbicides. Monsanto Study Number RPN-2007-227. January 2008.Monsanto Company 09-SY-201U Page 427 of 471 data to determine how a pesticide will move through and break down in the environment. Risks to birds, fish, invertebrates, mammals and plants are routinel y assessed and used in EPA’s determinations of whether a pestic ide may be licensed for use in the U.S.

EPA’s core pesticide risk assessment and regul atory processes ensure that protections are in place for all populations of nontarget spec ies. Because endangered species may need specific protection, EPA has developed risk assessment pr ocedures described in the Overview of the Ecological Risk Assessmen t Process (EPA, 2004a) to determine whether individuals of a listed species have the potential to be harmed by a pesticide, and if so, what specific protections may be appropria te. EPA’s conclusion regarding the potential risks a pesticide may pose to a listed species and any designated cri tical habitat for the species, after conducting a thor ough ecological risk assessme nt, results in an "effects

determination."

An assessment of the effects of glyphosate us e on all types of thre atened and endangered species was conducted by Monsanto. This a ssessment generally followed the procedures described in the Overview of the Ecologica l Risk Assessment Process (EPA, 2004a), as summarized in Figure O-1.Monsanto Company 09-SY-201U Page 428 of 471 Figure O-1. Tier I Endangered Species Assessment

Risk quotients (RQ’s) were calculated as the quotient of the EEC and the relevant toxicity endpoint for the most sensitive species for a given taxon (class of species). For acute studies of a few days duration, the concentr ation calculated to result in 50% mortality (LC 50 ) or 50% designated effect (EC 50 ) on the test species was utilized in the RQ calculation. For chronic studies, representi ng a significant portion of the species lifecycle, the highest concentrati on at which no effects were ob served (No Observed Effect Concentration, NOEC) was used in the RQ calculation.

Toxicity values (effects endpoi nts) for most categories of species were taken from the EPA assessment for new glyphosate uses on bentgrass (EPA, 2006a), or from EPA guideline studies conducted by Monsanto if th ese endpoints were lower. Studies from the literature were considered when the study design was appropriate for the assessment being made and where sufficient informati on regarding glyphosate or formulation test concentrations was available. Exposure es timates were based on standard EPA methods for calculating exposure (EPA, 2004a). Fo r aquatic organisms, the model GENEEC2 (EPA, 2004a), which calculates high-end estimates of surf ace water concentrations of pesticides in a generic farm pond, was utilized. When formulation toxicity was Exposure Estimate:

Worst Case

Estimated Environmental Concentration

(EEC) Using Standard EPA Models or

Anal y sis Methods Toxicity Endpoint (TE):

Lowest Acute or Chronic Value Meeting EPA Quality Standards

RQ ≥ Level of Concern (LOC)?

Further Analysis Determine Risk Quotient (RQ = EEC/TE)

Yes For Each

Taxon

Risk to Taxon Is Expected to be Low NoMonsanto Company 09-SY-201U Page 429 of 471 considered, default drift valu es and the EPA standard pond 38 were utilized for estimation of aquatic exposure. For terrestrial animals, the T-Rex model 39 was utilized to calculate estimated dietary exposure and risk. For terr estrial and semi-aquatic plants, only the drift component of the TerrPlant model (EPA, 2004a ) was used to determine exposure levels (the runoff component was disr egarded). Runoff was not cons idered to contribute to exposure, since glyphosate binds very tightly to agricultural soils and does not have herbicidal properties when bound to soil (EPA, 2006a).

The conclusion from this assessment, submitted to USDA is that threatened or endangered terrestrial or semi-aquatic pl ant species are not at risk from ground applications of glyphosate at ra tes less than 3.5 lb glyphosate (a.e.) per acre, or from aerial applications at rates less than 0.70 lb a. e. per acre. However, these species may be

at risk when rates exceed these levels. Sin ce the maximum single app lication rate before or after crop emergence in glyphosate-tolerant alfalfa is 1.55 lb a.e. per acre, no listed plant species are predicted to be at risk from ground application of glyphosate to glyphosate-tolerant alfalfa. Fo r other glyphosate-tolerant crop s, in-crop application rates are typically less than 3.5 lb a.e. per acre, re sulting in a prediction of no risk to listed plant species. Rates that exceed 3.5 lb a.e. per acre are generally for control of perennial species prior to crop emergence or prior to harvest.

The same assessment determined that other taxa (including birds, mammals, insects, fish, amphibians, aquatic invertebrates, and aquatic plants) were not at risk from the use of glyphosate herbicides in alfalfa production or in the production of other crops. Furthermore, this assessment determined that these other taxa were not at risk from indirect effects resulting from habitat alteration from th e use of glyphosate, since nonendangered terrestrial or semi-aquatic plants were not considered to be at risk of direct

effects.

Based on Monsanto’s determina tion that threatened and enda ngered plant species may be at risk from certain uses of glyphosate in cr op production (e.g., aeria l application), a more

detailed evaluation of the locations of threat ened and endangered plan t species relative to areas of crop production has been undertaken. The first crop to be assessed was alfalfa. The assessment process was divided into three phases, as outlined below.

o First, the co-occurrence of observations of threatened and endangered plant species and the presence of alfalfa pr oduction was determined at the county

level 40,41 . (Phase 1) This assessment considered the 2663 counties in which

38 A water body with a depth of 2 m and a volume of 20,000 liters.

39 U.S. Environmental Protec tion Agency. 2006. T-REX (Terrestrial Resi due EXposure Model). Version 1.3.1.

40 Priester T, Kemman R, Rives Frank A, Turner L, McGaughey B, Howes D, Giddings J, Dressel S. 2007. An

Analysis of Possible Risk to Threatened and Endangered Pl ant Species Associated with Glyphosate Use in Alfalfa: A County-Level Analysis. Monsan to Study Number CS-2005-125.

41 Priester T, Kemman R, Rives Frank A, Turner L, McGaughey B, Howes D, Giddings J, Dressel S. 2008. An

Analysis of Possible Risk to Threatened and Endangered Pl ant Species Associated with Glyphosate Use in Alfalfa: A County-Level Analysis (Supplement). Monsanto Study Number CS-2007-229.Monsanto Company 09-SY-201U Page 430 of 471 alfalfa is grown, which comprise 85% of the 3141 counties and equivalent areas 42

in the 50 states of the U.S.

o Next, in counties with both threatened or endangered plant species observations and alfalfa production, the possible exposur e of threatened and endangered plant species to glyphosate was assessed at the sub-county level. (Phase 2) This

assessment used information available at the sub-county level for threatened and endangered plant species locations and for la nd use. Land uses considered in this assessment are identified as Pasture/Hay and Cultivated Crops. 43

o Finally, in subcounty areas where, under certain a pplication conditions, the potential for threatened and endangered plant species to be at risk from exposure to glyphosate could not be excl uded, these areas have been defined so that grower practices can be implemented to limit glyphosate exposure. Measures to limit glyphosate exposure in these areas will be proposed. (Phase 3) These measures include (1) limiting ground application rates to less than 3.5 lb glyphosate a.e. per

acre in areas identified for potential use limitation when the potential habitat for the threatened or endangered species is present, and (2) for aerial applications implementing an unsprayed buffer between the potential habitat for the listed species and the application area. Buffers are proposed to be based on application rate, droplet size a nd wind direction.

This analysis has been comple ted for the 2663 U.S. counties in which alfalfa is grown. The evaluation of the remaining 478 counties of the U.S. is currently in progress, based on the presence of threatened or endangered plant species, the production of other crops in those counties, and the cultivated crop or pa sture/hay land use. In counties that have

observations of threatened a nd endangered plant species and agricultural land uses, a more detailed sub-county analysis will be conducted.

Of the 2663 U.S. counties where alfalfa is produced, less than 11 % of counties (284 counties) have required the defi nition of potential areas for use limitations. In the other 2379 counties, either there are no threatened or endangered plant species present, or the species present are either excluded from concern (based on habitat or proximity information), have existing protections, or ar e not in proximity to potential areas of alfalfa production that ar e not already excluded or protected in some way.

The Roundup Ready alfalfa assessmen t considered all land that could potentially be used for agricultural crop produc tion (in counties with report ed alfalfa farms) in the assessment of proximity to observations of threatened or endangered species. Thus, the identification of potential use limitation area s also applies to other crops in those

counties. Because alfalfa is grown in such a large number of counties, the endangered plant species assessment conducte d for alfalfa covers more th an 90% of the acres where corn, cotton, soybean, sugar be ets and canola are grown.

42 Equivalent areas include independent cities th at are not within the boundaries of a county.

43 Land use was based on the National la nd Cover Database (2001) for the con tinental U.S. and on the NOAA (National

Oceanic and Atmospheric Administration) Coastal Services Center land cover data for Hawaii.Monsanto Company 09-SY-201U Page 431 of 471 O.3.6 Potential Effects on Endangered Anim al Species Identified by EPA or in Litigation

As previously discussed, no indirect effect s on threatened or endangered animal species are predicted, since no si gnificant direct effects due to pesticide drift onto nonendangered plant species are predicted. In the Glyphosat e Reregistration Eligibility Decision (RED) (EPA, 1993), EPA suggested that glyphosate may have effects on the

habitat of the Houston Toad . After the issuance of th e 1993 Glyphosate RED, Monsanto conducted a vegetative vigor study. When re levant effects data from that study are considered, it can be determined that the amount of glyphosate per unit area predicted to drift away from the site of an agricultural application is le ss than the amount per unit area

observed to have a 25% effect on plant dry weight or growth of the most sensitive of ten species tested in the study. Thus, the habitat of the toad is not likely to be significantly affected by glyphosate drift, and hence the toad is not likely to be at risk from the agricultural use of glyphosate. Similar conclusions can be drawn with respect to the habitat of other endangered animal species, su ch as the California red-legged frog (which has been the subject of years-long litigat ion between public interest groups and the federal government 44 ). The assessment conducted also i ndicates that the frog itself is not at risk from aquatic exposures to glyphosat e used in agriculture because estimated exposure concentrations are much lower th an concentrations at which effects on amphibians have been observed.

The EPA also has evaluated the potential eff ect of glyphosate on salmon in eleven areas in California and Southern Oregon 45 in response to the consent agreement reached in

another lawsuit 46 ). The conclusion of the EPA’s risk assessment is as follows:

“For all uses with application rates of 5 lb a. i. per A or below, the Agency has determined that glyphosate will have No Effect on the subject listed species.” (EPA, 2004b,c). All glyphosate use rates for ag ricultural uses are 5 lb a.i. per acre (3.75 lb glyphosate a.e. per acre) or below, so no risk to salmon is anticipated from these uses.

O.3.7 Other Potential Environmental Impa cts Associated with Glyphosate Use in Glyphosate-tolerant Crops

As discussed more fully below, the potential im pacts to soil attributable to the change in production (cultivation) pr actices associated w ith the deregulation of glyphosate-tolerant crops have been assessed. The adoption of gl yphosate-tolerant crops and the ability to use glyphosate-based agricultural herbicides is not expected to significantly change agricultural practices, except to enable th e adoption of no-till s eeding practices.

1. No-Till Practices: No-till production is the practice of establishing an agricultural seed bed and controlling weed s without mechanically tillin g the soil. Instead, the only tillage of the soil is done at the time of plan ting, with the crop being seeded directly into the previous year’s crop residue. Among othe r environmental benefits, no-till production

44 Center For Biological Diversity v. Leavitt , 2005 WL 2277030 (N.D.Cal., September 19, 2005).

45 These areas are call Evolutionarily Significant Units ba sed on the salmonid populations present in these areas.

46

, Washington Toxics Coalition v. Environmental Protection Agency , 413 F.3d 1024 (9th Cir. 2005).Monsanto Company 09-SY-201U Page 432 of 471 reduces soil erosion and the use of petroleum- based fuels for tractors. The practice has

been shown to minimize surface water runoff and soil erosion and to improve soil quality by increasing the soil organic matter that helps bind soil nutrients and prevent their loss to runoff, erosion and leaching (Leep et al., 2003). Less soil erosion into surface waters would positively impact stream dynamics (McVay et al., 2005).

No-till agriculture can provide benefits to water bodies, as well. No-till practices reduce soil erosion to surface water bodies, decreasing the amount of sediment in rivers and

streams. Sedimentation increas es the turbidity (cloudiness) of surface water bodies, reducing light penetration, impairing phot osynthesis and altering oxygen levels, which

cause a reduction of food sources for some aquatic organisms. Sediment can also cover spawning beds and impact fish populations. P hosphorus (a major component of fertilizer) bound to soil particles can be transferred to rive rs and lakes via soil erosion, giving rise to high levels of phosphorus in su rface waters, which may lead to algae blooms that can impact desirable fish populations (Hill et al

. , 1995).

2. Soil Microorganisms : Soybean is a legume that forms a symbiotic relationship with the nitrogen-fixing bacterium Bradyrhizobiacea japonicum (see Section VIII.D.4). The effects of glyphosate and glyphos ate-based formulations on soil microorganisms have been extensively investigated (Sullivan and Su llivan, 2000). Results of standardized tests with glyphosate formulations performed for submission to re gulatory agencies indicate no long-term effects on microor ganisms in soil even at rates that exceed maximum use rates (up to five times the labeled rate). In addition, independe nt researchers have reviewed numerous laboratory and field studies, investiga ting the effects of glyphosate on soil bacteria and fungi 47 (Giesy et al., 2000). Although so me laboratory tests have shown effects on nitrogen-fixing bacteria (Moorma n et al., 1992; Santos and Flores, 1995) and soil fungi (Estok et al., 1989; Busse et al., 2001), effects are t ypically observed only under artificial laboratory conditi ons and at glyphosate concen trations well above normal field application rates. Several researchers have concluded that it is difficult to extrapolate results from the laboratory to th e natural soil environmen t (Estok et al., 1989; Wan et al., 1998; Busse et al., 2001).

In studying microorganisms from soil in pine plantations, Busse et al . (2001) state: “Our findings suggest that artifici al media assays are of lim ited relevance in predicting glyphosate toxicity to soil organi sms and that field rate applic ations of glyphosate should

have little or no affect on soil microbial co mmunities in ponderosa pine plantations.” Long-term studies following repeated applicati ons of Roundup agricult ural herbicides in the field for six (Olson and Lindwall, 1991) or over 10 years (Hart and Brookes, 1996; Biederbeck et al., 1997) have shown no det ectable adverse effects on soil microbes. Investigations by Haney et al. (2000, 2002) related to the increa sed use of glyphosatetolerant crops indicate that glyphosate was de graded over time by soil microbes, even at high application rates, without adversely impacting the so il microbial community. In

47 Felsot, A.S. 2001. Herbicide tole rant genes, Part 4: Withering wildlif e? Agric. & Environ News, No. 178. http://www.aenews.wsu.edu/ Feb01AENews/Feb01AENews.htm

.Monsanto Company 09-SY-201U Page 433 of 471 addition, results from field st udies that have evaluated th e fungal component of the soil microbial community indicate that glyphosate treatment had no deleterious effects on beneficial soil fungi (Araujo et al., 2003; Biederbeck et al., 1997; Busse et al., 2001; Wardle and Parkinson, 1990a,b). Moreover, the history of safe use and yield data obtained for nearly 10 years of glyphosate-tole rant crop production, combined with incrop applications of gl yphosate-based agricultural herbic ides, reinforce the findings that soil microbes and microbially mediated proc esses are not adversely impacted by fieldrate applications of glyphosate.

3. The Potential for Glyphosate Metal Chelation to Affect Soil Fertility: Plants are dependent on the uptake of a number of diffe rent metal cations from the soil for optimal growth. Glyphosate is known to chelate, or tight ly bind, to several di- and trivalent metal cations such as Fe3+, Cu2+, Mn2+, Al3+, and Ca2+ that are needed by plants (Madsen et al., 1978; Glass, 1984). Cations that chelate gly phosate have been shown to reduce the efficacy of glyphosate when present in su fficient amounts in the tank mix spray

solution 48 . In the spray solution, there is a simple interaction between glyphosate and metal cations, which reduces the herbicidal activ ity of glyphosate. Ho wever, in the soil environment, the interactions between meta ls and chelators are much more complex (Parker et al., 2005). Glyphosate can interact with metals th at are present on the surface

of soil particles, as well as with dissolved metal ions in the water soil solution. In addition to glyphosate, many other potential ligands or chelator s are present in soil that

can also interact with metals. As a result, there is a complex multi-component equilibrium between glyphosate, other ligands or chelators, and numerous metals present in soil. Glyphosate is only one factor in this system. Numerous compositional analysis studies have demonstrated a lack of any si gnificant immobilization of mineral nutrients by glyphosate in soil that results in reduced uptake by plants. These studies have shown that glyphosate-tolerant crops that have been sprayed with glyphosate do not have decreased micronutrient levels compared to untreated controls (McCann et al., 2006; Obert et al., 2004; Ridl ey et al., 2002).

4. Nitrogen Fixation: Nitrogen fixation is the proce ss by which inorganic nitrogen is fixed into an organic form nece ssary for plant growth. It oc curs in root nodules that are an integral part of the legume root system. The process of nitrogen fixation is subject to subtle variations depending on soil type and environmental conditions. While some laboratory investigations have indicated th at glyphosate may inhibit pure cultures of nitrogen-fixing bacteria (Moor man et al.1992; Santos and Flores, 1995), effects were only observed at glyphosate concentrations above normal field app lication rates.

Several researchers (King et al., 2001; Hoagland et al., 1999; Goos et al., 2002; Zablotowicz and Reddy, 2004) have investigated poten tial effects of glyphosate

formulated herbicides on nitr ogen-fixing bacteria associat ed with glyphosate-tolerant

48 de Ruiter, H., R.A. Downer, A.J.M. U ffing, T.A. Ebert, P.J.J. Pikaar and F. R. Hall. 2001. The influence of inorganic

cations on glyphosate activity - review and perspectives. American Society for Testing and Materials,

West Conshohocken.Monsanto Company 09-SY-201U Page 434 of 471 soybean. In general, any effects observed on the formation of nodules or nitrogen fixation were not observed uniformly and were noted to be transient in nature. Hoagland et al. (1999) reported some reduction in nodul ation in Roundup Ready soybean, but noted that effects were of minimal consequence due to the soybean’s ability to compensate for short durations of stress caused by enviro nmental factors such as high or low temperature, water availability, or nutrient status. King et al. (2001) reported that application of Roundup Ultra ® herbicide delayed nitrogen fixation and decreased nitrogen accumulation in some glyphosate-tole rant soybean cultivars. However, effects were only observed under droug ht conditions and at rate s of glyphosat e above the recommended label rates. The soybean yield was not affected. In a study which included four soybean varieties and four sites, G oos et al. (2002) reported that there was no indication Roundup Ultra herbicide inhibited nitrogen fi xation, except that in one soybean variety at one site there was a small reduction in ureides, th e principal products of nitrogen fixation in shoots of soybean. At recommended us e rates, the application of glyphosate-based herbicides in the glyphosate- tolerant soybean produc tion system is not expected to negatively affect soil fertility, nodule formation, or nitrogen fixation.

5. Transport through the Soil – Surfactant: Available data also suggest that the POEA surfactant used in Roundup agricu ltural herbicides binds str ongly to soil (estimated soil organic carbon-water parti tion coefficient (Koc) valu es range from 2500 to 9600 49 ) and undergoes microbial degradati on with an estimated half- life of less than 14 days 50

. POEA is rapidly partitioned (h alf-life of 13 to 18 hours) from water to sediment in a water / sediment study (Wa ng et al., 2005). Th e rapid partitioning of the POEA

surfactant to soil sediment combined with the high Koc values indicates that the surfactant will be tightly bound to the soil. Th e Groundwater Ubiquity Score, GUS, is an index that indicates the potential for com pounds to leach from soil into groundwater,

based on their half-life and Ko c (Gustafson, 1989). Using an estimated half-life of 14 days and a Koc of 2500 as conservative estimat es of the rate of degradation and binding to soil, the GUS index for the POEA surfactan t is 0.69. According to the GUS movement ranking, this GUS index indicates that PO EA has a very low potential to leach to groundwater.

® Roundup Ultra is a resistered trad emark of Monsanto Technology LLC.

49 Estimated from the partition ratio between water and sterile soil as reported in the POEA soil degradation study,

Marvel et al., 1974.

50 Marvel, J.T., Brightwell, B.B. and Suba, L.A. 1974. G3780A Surfact ant: Biodegradation, plant uptake and 14 C

Distribution. Monsanto Company Report No. 321Monsanto Company 09-SY-201U Page 435 of 471 Attachment 1. Appendix O. U.S. Glyphosate Tolerances for Food & Feed Commodities (40 CFR § 180.364)

Commodity Parts/million Commodity Partss/million

Acerola 0.2 Durian 0.2

Alfalfa, seed 0.5 Egg 0.05

Almond, hulls 25 Epazote 1.3

Aloe vera 0.5 Feijoa 0.2

Ambarella 0.2 Fig 0.2

Animal feed, nongrass, group 18 400 Fish 0.25

Artichoke, globe 0.2 Flax, meal 8.0

Asparagus 0.5 Flax, seed 4.0

Atemoya 0.2 Fruit, citrus, group 10 0.5

Avocado 0.2 Fruit, pome, group 11 0.2

Bamboo, shoots 0.2 Fruit, stone, group 12 0.2

Banana 0.2 Galangal, roots 0.2

Barley, bran 30 Ginger, white, flower 0.2

Barley, grain 20 Goat, kidney 4.0

Beet, sugar, dried pulp 25 Goat, liver 0.5

Beet, sugar, roots 10 Gourd, buffalo, seed 0.1

Beet, sugar, tops 10 Governor's plum 0.2

Berry group 13 0.2 Gow kee, leaves 0.2

Betelnut 1.0 Grain, as pirated fractions 100

Biriba 0.2 Grain, cereal, forage, fodder and straw, group 16, except corn forage 100

Blimbe 0.2 Grain, cereal, group 15, except barley, field corn, grain sorghum, oat and wheat 0.1

Borage, seed 0.1 Grape 0.2

Breadfruit 0.2 Grass, forage, fodder and hay, group 17 300

Cacao bean 0.2 Guava 0.2

Cactus, fruit 0.5 Herbs subgroup 19A 0.2

Cactus, pads 0.5 Hog, kidney 4.0

Canistel 0.2 Hog, liver 0.5

Canola, meal 15 Hop, dried cones 7.0

Canola, seed 10 Horse, kidney 4.0

Cattle, kidney 4.0 Horse, liver 0.5

Cattle, liver 0.5 Ilama 0.2

Chaya 1.0 Imbe 0.2

Cherimoya 0.2 Imbu 0.2

Citrus, dried pulp 1.5 Jackfruit 0.2

Coconut 0.1 Jaboticaba 0.2

Coffee, bean 1.0 Jojoba, seed 0.1

Corn, field, forage 6.0 Juneberry 0.2

Corn, field, grain 1.0 Kava, roots 0.2

Cotton, gin byproducts 175 Kenaf, forage 200

Cotton, undelinted seed 35 Kiwifruit 0.2

Cranberry 0.2 Lesquerella, seed 0.1

Crambe, seed 0.1 Leucaena, forage 200

Custard apple 0.2 Lingonberry 0.2Monsanto Company 09-SY-201U Page 436 of 471 Commodity Parts/million Commodity Parts/million

Date 0.2 Longan 0.2

Dokudami 2.0 Lychee 0.2

Mamey apple 0.2 Sapote, black 0.2

Mango 0.2 Sapote, mamey 0.2

Mangosteen 0.2 Sapote, white 0.2

Marmaladebox 0.2 Sesame, seed 0.1

Meadowfoam, seed 0.1 Sheep, kidney 4.0

Mioga, flower 0.2 Sheep, liver 0.5

Mustard, seed 0.1 Shellfish 3.0

Noni 0.20 Sorghum, grain, grain 15

Nut, pine 1.0 Soursop 0.2

Nut, tree, group 14 1.0 Soybean, forage 100

Oat, grain 20 Soybean, hay 200

Okra 0.5 Soybean, hulls 100

Olive 0.2 Soybean, seed 20

Oregano, Mexican, leaves 2.0 Spanish lime 0.2

Palm heart 0.2 Spearmint, tops 200

Palm heart, leaves 0.2 Spice subgroup 19B 7.0

Palm, oil 0.1 Star apple 0.2

Papaya 0.2 Starfruit 0.2

Papaya, mountain 0.2 Stevia, dried leaves 1.0

Passionfruit 0.2 Strawberry 0.2

Pawpaw 0.2 Sugar apple 0.2

Pea, dry 8.0 Sugarcane, cane 2.0

Peanut 0.1 Sugarcane, molasses 30

Peanut, hay 0.5 Sunflower 85

Pepper leaf, fresh leaves 0.2 Sunflower, seed 0.1

Peppermint, tops 200 Surinam cherry 0.2

Perilla, tops 1.8 Tamarind 0.2

Persimmon 0.2 Tea, dried 1.0

Pineapple 0.1 Tea, instant 7.0

Pistachio 1.0 Teff, grain 5.0

Pomegranate 0.2 Ti, leaves 0.2

Poultry, meat 0.1 Ti, roots 0.2

Poultry, meat byproducts 1.0 Ugli fruit 0.5

Pulasan 0.2 Vegetable, leafy, brassica, group 5 0.2

Quinoa, grain 5.0 Vegetable, bulb, group 3 0.2

Rambutan 0.2 Vegetable, cucurbit, group 9 0.5

Rapeseed, meal 15 Vegetable, foliage of legume, except soybean, subgroup 7A 0.2

Rapeseed, seed 10 Vegetable, fruiting, group 8 0.1

Rose apple 0.2 Vegetable, leafy, except brassica, group 4 0.2

Safflower 85 Vegetable, leaves of root and tuber, group 2, except sugar beet 0.2

Safflower, seed 0.1 Vegetable, legume, group 6, except soybean 5.0

Salal 0.2 Vegetable, legume, group 6 except soybean and pea,dry 5.0Monsanto Company 09-SY-201U Page 437 of 471 Commodity Parts/million Commodity Parts/million

Sapodilla 0.2 Vegetable, root and tuber, group 1, except sugar beet 0.2

Wasabi, roots 0.2 Wheat, grain 5.0

Water spinach, tops 0.2 Wheat, middlings 20

Watercress, upland 0.2 Wheat, shorts 20

Wax jambu 0.2 Yacon, tuber 0.2

Wheat, bran 20Monsanto Company 09-SY-201U Page 438 of 471 Appendix O References

Acquavella, J.F., B.H. Alexander, J.S. Mandel, C. Gustin, B. Baker, P. Chapman, M. Bleeke. 2004. Glyphosate biomonitoring for farmer-applicators and their families: Results from the farm family exposure study. Environmental Health Perspective 112:321-

326.

Araujo, A.S.F., R.T.R. Monteiro, and R.B. Abarkeli8. 2003. Effect of glyphosate on the microbial activity of two Brazili an soils. Chemosphere 53:799-804.

Ashton, F.M. and A.S. Crafts. Glyphosate. 1981. Pages 243-244 in Mode of Actions of Herbicides. John Wiley & Sons, New York.

Biederbeck, V.O., C.A. Campbell, and H. J. Hunter. 1997. Tillage effects on soil

microbial and biochemical characteristics in a fallow-wheat rotation in a dark brown soil.

Canadian Journal of Soil Science 77:309-319.

Busse, M.D., A.W. Ratcliff, C.J. Shestak, and R.F. Powers. 2001. Glyphosate toxicity and the effects of long-term vegetation c ontrol on soil microbial communities. Soil Biology and Biochemistry 33:1777-1789.

EPA. 2006a. Glyphosate new use (bent-grass). U.S. Envir onmental Protection Agency

Environmental Fate and Effects Risk Asse ssment DP D3234409, Washington, D.C.

EPA. 2006b. Glyphosate human heal th risk assessment for proposed uses on safflower

and sunflower. Petition 4E6878 DP 314476. U. S. Environmental Protection Agency, Washington, D.C.

EPA. 2006c. Glyphosate human health risk as sessment for proposed use on Indian mulberry and amended use on pea, dry. Pe tition 5E6987 DP 321992. U.S. Environmental Protection Agency, Washington, D.C.

EPA. 2006d. Glyphosate; Pesticid e tolerance. U.S. Envir onmental Protection Agency, Washington, D.C. 71 FR 76180.

EPA. 2004a. Overview of the ecological ri sk assessment process in the Office of Pesticide Programs. U.S. Environmental Pr otection Agency, Endangered and Threatened Species Effects Determinations, Washington, D.C.

EPA. 2004b. Glyphosate analysis of risks to endangered and threatened salmon and steelhead. http://epa.gov/espp/ litstatus/effects/gly phosate-analysis.pdf [Accessed July 2, 2009].

EPA. 2004c. Effects dewtermination fo r glyphosate on Pacific salmonid ESUs. Transmittal letter from Williams, A-J B. Chief, Environmental Field Branch to L. Allen, Acting Director, Office of Prot ected Resources, National Marine Fisheries Service. U.S. Environmental Protection Ag ency, Washington, D.C.

EPA. 2004d. Glyphosate; Pesticid e tolerance. U.S. Envir onmental Protection Agency, Washington, D.C. 69 FR 65081.

EPA. 2003. Glyphosate; Pesticide tolerance. U.S. Environmental Protection Agency, Washington, D.C. 68 FR 36472.Monsanto Company 09-SY-201U Page 439 of 471 EPA. 2002. Glyphosate; Pesticid e tolerances. U.S. Environmental Protection Agency, Washington, D.C. 67 FR 60934.

EPA. 2000. Glyphosate; Pesticide tolerance. U.S. Environmental Protection Agency, Washington, D.C. 65 FR 57957.

EPA. 1997. Glyphosate; Pesticid e tolerances. U.S. Environmental Protection Agency, Washington, D.C. 62 FR 17723.

EPA. 1999. Glyphosate; Pesticid e tolerances. U.S. Environmental Protection Agency, Washington, D.C. 62 FR 17723.

EPA. 1996a. Pesticide tolerance for glyphosate. U.S. Environmental Protection Agency, Washington, D.C. 61 FR 7729.

EPA. 1996b. Pesticide tolerances for glyphosate. , U.S. Environmental Protection Agency, Washington, D.C. 61 FR 15192.

EPA. 1993. Reregistration elig ibility decision (R.E.D.) glyphosate. EPA 738-R-93-014,

U.S. Environmental Protection Agency, Washington, D.C.

Estok, D., B. Freedman, and D. Boyle. 1989. Effects of the herbicides 2,4-D, glyphosate, hexazinone, and triclopyr on th e growth of three species of ectomycorrhizal fungi.

Bulletin of Environmental Contam ination and Toxicology 42:835-839.

Giesy, J.PO., S. Dobson, and K.R. Solomon. 2000. Ecotoxicologica l risk assessment for Roundup herbicide. Rev. Environ. Contamn. Toxicol. 167:35-120.

Glass, R.L. 1984. Metal complex formation by glyphosate. Journal of Agricultural Food and Chemistry 32:1249-1253.

Goos, R.J., B. Johnson, M. Reinert, T. Helms, R. Henson, and L. Martin. 2002. Effect of Roundup on nitrogen fixation by Roundup Ready soybeans. Proceedings of the Great Plains Soil Fertility Conference.

Gustafson, D.I. 1989. Groundwater ubiquity score: A simple me thod for assessing pesticide leachability. Environmenta l Toxicology and Chemistry 8:339-357.

Haney, R.L., S.A. Senseman, F.M. Hons, a nd D.A. Zuberer. 2000. Effect of glyphosate on soil microbial activity and biom ass. Weed Science 48:89-93.

Haney, R.L., S.A. Senseman, and F.M. Hons. 2002. Effect of Roundup Ultra on microbial activity and biomass from selected soils. Journal of Environmental Quality 31(3):730-735.

Hart, M.R. and P.C. Brookes. 1996. Soil micr obial biomass and mineralization of soil organic matter after 19 years of cumulative fi eld applications of pesticides. Soil Biology and Biochemistry 28:1641-1649.

Hill, P.R. and J.V. Mannering. 1995. Conser vation tillage and water quality. Purdue University Cooperative Extensi on Service Publication WQ20. http://www.ces.purdue.edu/extmedia/WQ/ WQ-20.html [Accessed July 2, 2009].

Hoagland, R.E., K.N. Reddy, and R.M. Za blotowicz. 1999. Effects of glyphosate on Bradyrhizobium japonicum interactions in Roundup Ready soybeans. Annual Meeting of the Weed Science Society of American, Vol. 39.Monsanto Company 09-SY-201U Page 440 of 471 Kiely, T., D. Donaldson, and A. Grube. 2004. Pesticide industry sales and usage: 2000

and 2001 market estimates. Bi ological and Economic Anal ysis Division, Office of Pesticide Programs, U.S. Environmenta l Protection Agency, Washington, D.C.

King, C.A., L.C. Purcel, and E.D. Vories. 2001. Plant growth and nitrogenase activity of glyphosate-tolerant soybeans in response to foliar glyphosate applications. Agronomy

Journal 93:179-186.

Leep, R., D. Undersander, P. Peterson, D-H Min, T. Harrigan, and J. Grigar. 2003. Steps to successful no-till establishment of forages. Michigan State University, University of Wisconsin, University of Minnesota, and Na tural Resources Cons ervation Service,

Extension Bulletin E-2880.

Madsen, H.E.L., H.H. Christensen, and C. Go ttlieb-Petersen. 1978. Stability constants of copper (II), zinc, manganese (II), calcium, and magnesium complexes of N- (phosphonomethyl) Glycine (glyphosate). Acta Chemica Sca ndinavica A 32:79-83.

McCann, M.C., G.J. Rogan, S. Fitzpatrick, W. A. Trujillo, R. Sorbet, G.F. Hartnell, S.G. Riordan, and M.A. Nemeth. 2006. Glyphosat e-tolerant alfalfa is compositionally equivalent to conventional alfalfa (Medicago sativa L.). Journal of Agriculture and Food Chemistry 54:7187-7192.

McVay, K.A., D.L. Levlin, and J. Neel. 2005. Effects of conservation practices on water quality: Sediment. Kansas State Universi ty Agricultural Experiment Station and Cooperative Extension Service MF2682.

Moorman, T.B., J.M. Becerril, J. Lydon, and S.O. Duke. 1992. Production of hydroxybenzoic acids by Bradyrhizobium ja ponicum strains after treatment with glyphosate. Journal of Agricultura l and Food Chemistry 40:289-293.

Obert, J.C., W.P. Ridley, R.W. Schneider, S.G. Riordan, M.A. Nemeth, W.A. Trujillo, M.L. Breeze, R. Sorbet, and J.C. Astwood. 2004. The composition of grain and forage from glyphosate-tolerant wheat MON 71800 is e quivalent to that of conventional wheat (Triticum aestivum L.). Journal of Ag ricultural and Food Chemistry 53:1375-1384.

Olson, B.M. and C.W. Lindwall. 1991. So il microbial activity under chemical fallow conditions: Effects of 2,4-D and glyphosate. Soil Biology and Biochemistry 23(11):1071-

1075.

Parker, D.R., S.M. Reichman, and D.E. Crowley. 2005. Metal chelation in the rhizosphere. Pages 57-93 in Roots and So il Management: Interactions Between Roots and the Soil. American Society of Agronomy, Madison, Wisconsin.

Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. Comparison of the nutritional profile of glyphos ate-tolerant corn event NK603 with that of conventional corn (Zea mays L.). J ournal of Agricultural and Food Chemistry

50:7235-7243.

Santos, A. and M. Flores. 1995. Effects of glyphosate on nitrogen fi xation of free-living heterotrophic bacteria l. Letters in Applied Microbiology 20:349-352.

Sullivan, D.S. and T.P. Sullivan. 2000. Non-target impacts of the herbicide glyphosate: A compendium of references and abstracts. Applied Mammal Research Institute, Summerland, British Columbia.Monsanto Company 09-SY-201U Page 441 of 471 Wan, M.T., J.E. Rahe, and R.G. Watts. 1998. A new technique for determining the sublethal toxicity of pestic ides to the vesicular-arbuscu lar mycorrhizal fungus Glomus

intraradices. Environmental T oxicology Chemistry 17(7):14-21.

Wang, N., J.M. Besser, D.R. Buckler, J.L. H onegger, C.G. Ingersoll, B.T. Johnson, M.L. Kurtzweil, J. MacGregor, and M.J. McKee. 2005. Influence of sediment on the fate and toxicity of a polyethoxylate d tallowamine surfactant system (MON 0818) in aquatic microcosms. Chemosphere 59:545-551.

Wardle, D.A. and D. Parkinson. 1990a. Infl uence of the herbicid e glyphosate on soil microbial community structur e. Plant and Soil 122:21-28.

Wardle, D.A. and D. Parkinson. 1990b. Eff ects of three herbicides on soil microbial biomass and activity. Plant and Soil 122:29-37.

WHO/FAO. 2004. Pesticide residues in food — 2004. Report for the Jo int Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Envi ronment and the WHO Core Assessment Group on Pesticide Residue s (JMPR). Rome, September 20-29, 2004.

FAO Plant Production and Protec tion Paper 178. World Health Organization and United Nations Food and Agricultu ral Organization, Rome.

Zablotowicz, R.M. and K.N. Reddy. 2004. Im pact of glyphosate on the Bradyrhizobium japonicum symbiosis with glyphosate-resist ant transgenic soyb ean: A Minireview. Journal of Environmental Quallity 33:825-831.Monsanto Company 09-SY-201U Page 442 of 471 Appendix P. Petitioner’s Environmental Assessment

P.A. Background

USDA-AHPIS has responsibility, under the Plant Protection Act (7 U.S.C. § 7701-7772), to prevent the introduction and dissemination of plant pests into the U.S. Additionally, USDA-APHIS must comply with the Nationa l Environmental Policy Act (NEPA) when making the decision whether to grant deregulat ed status to MON 87705 . Numerous field

trials conducted in the U.S. under APHIS notifications and permits since 2005 have

included MON 87705 as test material. Information has been collected from these field trials, other tests, and the literature to assess whether the improved fatty acid profile through suppression of FATB and FAD2 RNAs, expression of the CP4 EPSPS enzyme to confer glyphosate tolerance and/or the plant transformation process has altered MON 87705 in any way that woul d impart plant pest characte ristics or caus e significant environmental impacts, includi ng cumulative impacts. The purpose of this section is to provide relevant, trait-specific informati on regarding the potential for reasonably foreseeable, significant environmental impacts.

An analysis of the potential impact of deregulation of MON 87705 on current soybean agronomic production systems, and related acti vities such as soybean processing, food and feed uses as well as marketing of soybean and soybean products is presented in this section. Factors evaluated as part of th e assessment include potential impacts to:

• land use patterns, non-agricultural la nds, farming practices, commodity and specialty soybean production,

• marketability of soybean seed for planti ng and seed for specialty and commodity markets, and

• public health, non-target organisms, th reatened or endangered species, and biodiversity. The analysis conducted considers current cond itions, the potential for deregulation of MON 87705 to impact these conditions, and poten tial cumulative impacts. In most cases, there are no impacts to current conditions (e .g., no differences be tween deregulation of MON 87705 versus continuing to regulate). Where diffe rences were noted, these

differences are described and th eir significance evaluated.

P.B. Purpose and Need

Monsanto Company (Monsanto) is submitting to APHIS this petition for the determination of nonregulated status for MON 87705 plants genetically enhanced to suppress the endogenous FATB and FAD2 RNA in the developing soybean seed; thereby improving the fatty acid profile to contain lo wer saturated fat, hi gher oleic, and lower polyunsaturated fatty acid le vels. MON 87705 soybean oil also has improved heat and oxidative stability and, due to the lower levels of saturated fats, a healthier profile relative to conventional soybean oil. MON 87705 also expresses CP4 EPSPS protein throughout the plant conferring tolerance to glyphosate, which is the activ e ingredient in the Roundup family of agricultural herbicides.

Initially the U.S. market for soybean oil was severely limited by the presence of

undesirable off-flavors and odor s. The double bonds in the polyunsaturated fatty acidsMonsanto Company 09-SY-201U Page 443 of 471 (PUFAs), particularly linolenic acid, are susceptible to oxidation resulting in the formation of “fishy” or acrid flavors and odors in soybean o il (Dutton et al., 1951). As a result, chemical hydrogenation was adopted to reduce the cont ent of polyunsaturated fatty acids (Dutton, 1963; Okkerse et al., 1967). At first, hydrogena ted vegetable oils, including hydrogenated soybean oil, were viewed as viable alte rnatives to animal fats and the high-in-saturated-fats tropi cal oils such as palm oil and coconut oil. However, in the 1990s, nutrition research showed that the trans fatty acids in hydroge nated oils created by the hydrogenation process had negative health consequences (Judd et al., 1994; Mensink and Katan, 1990; Zock and Katan, 1992). By 2006, the U.S. FDA had issued a regulation obligating food manufactu rers to declare the trans fatty acid content of their product on nutrition labeling (FDA, 2006). As a result, soybean oil per capita consumption in U.S. has been declining since 2006 (Soyatech, 2008).

MON 87705 soybean oil fatty aci d profile provides important new formulation options for food companies interested in the developm ent of lower saturate d fat food products to support heart health. Because MON 87705 soybean oil has a reduced level of PUFAs, it has higher oxidative stability without the need for hydrogenati on, and has lower levels of saturated fats compard to commodity soyb ean. Saturated fats, notably palmitic acid (16:0), contribute to cardiovascular disease a nd other chronic diseases in epidemiological trials (Hu et al., 1997). As a result, reduci ng saturated fat levels in soybean oil can positively impact the goal of keeping human dietary consumption of saturated fats below 10% of their total energy intake (USHHS, 2005).

As mentioned, a reduction in saturated fats and increa sed oxidative stability in MON 87705 soybean oil contribu te to an important new formulation option for food companies. The uses of soybean oil for biodies el and other industrial applications is also enhanced by virtue of a m odified oil profile similar to that found in MON 87705 soybean oil. Low saturated fats and high (>70%) olei c acid levels are key at tributes for vegetable oils targeted for biodiesel and industrial us es because these characteristics are vital to improved cold weather performance, impr oved stability, and re duced nitrous oxide emissions (Knothe, 2005; Graef et al., 2009).

Therefore, the decrease in sa turated fats and PUFAs (17% vs. 60% FA), in MON 87705 soybean oil provides important options for f ood companies to develop lower saturated fat foods with greater food functionality. In addition, MON 87705 soybean oil attributes provide key enhancements for biodies el and industrial applications.

P.C. Soybean Production

The following section describes the setting for the proposed deregulation and provides the context for evaluating the intensity of the impact due to USDA-APHIS granting deregulated status to MON 87705. The proposed deregulation would be relevant to the production of an intensively cultivated row crop - soybean . Soybean is grown as a commercial crop in over 35 count ries. In the United States, it is generally grown on greater than 70 million acres in at least 27 states with over a million acres grown in each of the following states: IA, IL, MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, NC, KY, TN (USDA-NASS, 2006).

In recent years, there has been an increased demand by c onsumers and food processers for soybean and other oilseed crops that have specific physical or ch emical characteristicsMonsanto Company 09-SY-201U Page 444 of 471 to meet specific food or feed needs. Appr oximately 12% of soybean grown is specialty soybean produced for a specific market or use (United Soybean Board at http://www.unitedsoybean.org), including hi gh protein, tofu, nonbi otechnology-derived, organic and high oleic and lo w linolenic acid content. The specialty, value-added product may be the whole bean or a fraction such as the oil. The vast majority of soybean in the U.S. is grown for animal feed and is fed as soybean meal. During processing, soybean oil is extracted to produce crude soybean oil and defatted soybean meal. According to a 1999 American Soybean Asso ciation sponsored study the oil content of soybean meal after extraction is relatively low, between 1-3%. (http://www.soymeal.org/worldlitart icles_new/globalsmsampling.html ).

Specialty soybean varieties are specified by buyers and end-users of soybean for production, and premiums are paid for deliver ing a product that meet s purity and quality standards for the soybean variety. Product di fferentiation and market segmentation in the specialty soybean industry includes mech anisms to keep track of the soybean (traceability), methods for identity preser vation (IDP), such as quality assurance processes (e.g., ISO9001-2000 cert ification), as well as cont racts between growers and buyers that specify delivery agreements. U ltimately, the amount of planted acreage will be driven by consumer preferences based on the demand for the oil produced by MON 87705. On the basis of predicted dema nd for food and feed applications, and the additional health benefits and functionality of MON 87705 soybean oil, it is initially expected to be a specialty soybean produced for its premium oil. This premium is determined by the increased cost of produc tion and distribution throughout the supply chain (mostly IDP costs) and market demand for the enhanced composition, which in the case of MON 87705 soybean oil is an improved fa tty acid profile that is higher in oleic acid and lower in saturated fats than commodity soybean oil

Data in Section VII of this petition show that, aside from the improved fatty acid profile present in the oil, the other processed fractions (meal, lecith in, and protein isolate) from MON 87705 seed are compositionally and nutri tionally equivalent to nonbiotechnologyderived conventional processed fractions.

This deregulation is being sought in an environment that has rapidly adopted biotechnology-derived soyb ean varieties (James, 2007). Thirteen different biotechnology-derived soybean events have been granted nonregulat ed status by USDA since 1994 (www.aphis.usda.gov). In pa rticular, biotechnology- derived herbicidetolerant soybean varieties were grown on approximately 69 million of the 75 million acres of soybean grown in the U.S. in 2008 (USDA-NASS, 2008). Thus, soybean breeders, seed manufactures, and soybean producers have developed practices and systems that allow for the concurrent br eeding and manufacture of biotechnology-derived and specialty soybean and are capable of breeding and manuf acturing seed, and producing harvested seed to meet th e needs of various markets.

Traditional plant breeding has be en used to alter the fatty ac id profiles of oil producing plants such as soybean. For example, soybean varieties with lower levels (1-3%) of the polyunsaturated linolenic fatty acid have recently entered the market (Fehr, 2007) and programs to develop high oleic soybean thr ough conventional breeding have resulted in soybean varieties with an ol eic content of >70% (Alt et al., 2005). More recently soybeans with alterations in the fatty acid prof ile from genetic modifications have either been submitted to or deregulated by USDA-APHIS (Table P-1).Monsanto Company 09-SY-201U Page 445 of 471 Table P-1. Deregulated or Submitted Biotechnology-derive d Soybean Products with

Altered Oil Profiles

Phenotype ID Code(s) Institution Date Deregulated

Altered Oil Profile MON 87769 Monsanto Submitted

High Oleic Acid DP-3Ø5423-1 Pioneer Submitted Altered Oil Profile G94-1, G94-19, G-168 DuPont May, 1997

Source: http://www.aphis.us da.gov/brs/not_reg.html

The MON 87705 soybean oil improved fatty acid profile provide s important new formulation options for food companies interest ed in the development of lower saturated fat food products to support heart health. C onventional soybean oil typically contains 60- 65% polyunsaturated fatty acids. Because M ON 87705 soybean oil has a reduced level of polyunsaturated fatty acids, it has higher oxidative stabili ty without the need for hydrogenation, and has lower levels of saturated fats compard to commodity soybean. The low saturated fat/high oleic oil produced from MON 87705 soybeans is suitable for use in products such as vegetable oils, sala d dressings, margarine, etc., or in other applications for which commodity soybeans ar e used. Additionally, the use of soybean

oil for biodiesel and other i ndustrial applications also is enhanced by virtue of an improved fatty acid profile found in MON 87705 soybean oil.

P.D. Potential Environmental Impacts

The soybean production environment and MON 87705 are described in detail throughout this petition. The discussion of impacts below is focused on specific areas of the production environment and related activi ties including land use patterns, farming practices, commercial activitie s such as seed production an d marketing of soybean and impacts to the quality of agricultural products.

P.D.1. Impacts on Land Use

S oybean is grown as a commercial crop on over 75 million acres in at least 27 states in the U.S. (USDA-NASS, 2009a). Soybean acreage in the past fi ve years has been relatively stable varying from 64.7 million to 75.7 million acres with a 10-year average of 73.3 million acres (Table IX-1). Fluctuations in soybean acreage are due to environmental, agronomic and economic factors 1 , as well as government pr ograms such as the crop reserve program (CRP) or ethanol mandates imposed by the U.S. government. Soybean fields are typically highly mana ged agricultural areas that can be expected to be dedicated to crop production for many years and cul tivation of MON 87705 is not expected to differ from typical soybean cultivation practic es. The improved fatty acid profile trait provides growers with an optio n to produce a value added so ybean and potentially greater profitability from their farming operation (see Section IX). A dditionally, canola and

1 2008 NASS crop acreage report [http://www.nass.usda.gov/Newsroom/2008/06_30_2008.asp ] accessed

6/2009Monsanto Company 09-SY-201U Page 446 of 471 sunflower production acreage has fluctuated due to complex market demands. During the period of 1998 through 2008, canola acreag e has ranged from 865,000 to 1,555,000 acres. Likewise, during the same period, s unflower acreage fluctuated between 246,869 and 669,338 acres 2 . MON 87705 will likely be used in common rotations on land previously used for agricultural purposes.

There is no indication that the introduction and widespr ead adoption of biotechnologyderived crops in general has resulted in a significant change to the total U.S. acreage devoted to agricultural produc tion. The cumulative land ar ea in the U.S. planted to principal crops, which include corn, sorghum, oats, barley, winter wheat, rye, durum, spring wheat, rice, soybean, peanuts, sunflo wer, cotton, dry edib le beans, potatoes, canola, proso millet, and sugar beets, has rema ined relatively constant over the past 25 years. From 1983 to 1995, the average yearly acreage of principal crops was 328 million. This average is statistically unchanged at 326 million acres since the introduction of biotechnology-derived crops in 1996 3

. Granting deregulated status to MON 87705 is not expect ed to significantly alter commercial soybean cultivation in terms of agricultural i nputs and production management (see Section IX). Currently, biotechnology-derived herbicide tolerant soybean is planted on 92% of the soyb ean acreage (USDA-NASS, 2008) and the Roundup Ready soybean system has become the standard weed control program in U.S. soybean production. Therefore the presen ce of the herbicide tolerance trait in

MON 87705 does not offer a new or different incentive to growers that would be expected to significantly alter commercial soybe an cultivation.

A possible cumulative impact from the in troduction of herbicide-tolerant soybean

varieties is the increase in the practice of no-till or conservati on tillage by U.S. growers. In 1995, before the introduction of glyphosate- tolerant soybeans, approximately 27% of the U.S. soybean acres used no-till production. By 2004, no-till acres increased to 36% of the total soybean acres (Sankula, 2006). A few states provide statistics on the adoption

of no-till acres in their state. In 2006 in Illinois, no-till farming was used on 51% of the soybean acres. The University of Illinois Extension Service attributed that figure to the fact that 90% of the state’s acres were plan ted to glyphosate-tolerant varieties, along with other factors, such as high fuel prices, im proved equipment, higher yields using no-till practices, and better grower awareness of the advantages to soil and water quality from no-till farming 4 . For an overview of the cumulativ e impacts (includi ng tillage) on land use from deregulation of glyphosate-to lerant crops see Appendix N.

Additionally, because MON 87705, with the exce ption of the improved fatty acid profile, is phenotypically and ag ronomically equivalent to commerci al soybean varie ties, there is no expectation that introduction of MON 87705 will significantly alter the range of commercial soybean cultivation. Although it is not known how many acres will be planted to MON 87705, it is expected that th is product and/or subsequent combinations

2 Comparisons of canola and sunflower acreage established from databases at http://usda.mannlib.cornell.edu/usda/current/htrcp/

3 Calculated from database at http://usda.mannlib.cornell.edu/usda/current/htrcp by comparing the total

acres from 1983 to 1995 and from 1996 to 2007.

4 University of Illinois Extension Se rvive News Release: No-till is now the "Conventional" Tillage System for Illinois Farmers; http://web.extension.u iuc.edu/state/newsdetail.cfm?NewsID=4991Monsanto Company 09-SY-201U Page 447 of 471 of this product with other biotechnology-de rived or conventiona l soybean varieties potentially could be grown on the majo rity of U.S. soybean acres.

Because of the similar oil profiles, it is possible that the in troduction of MON 87705 and/or subsequent combinations of M ON 87705 with other bi otechnology-derived and conventional soybean varieties could impact de mand for oil from other oilseed crops such as canola and sunflower. The decision to pl ant a crop is based on many factors including demand, input costs, environmental conditi ons and geography. S oybean acreage does not tend to overlap land typically used for ca nola and sunflower production in the U.S. (Figure P-1). Additionally, canola and sunflower production acreage has fluctuated due to complex market demands. During the period of 1998 through 2008, canola acreage has ranged from 865,000 to 1,555,000 acres . Likewise, during the same period, sunflower acreage fluctuated between 246,869 and 669,338 acres 5 . Because of these geographic differences and complex market dyna mics, it is unlikely that the introduction of MON 87705 will significantly impact the planting of other oil producing crops and

should not result in si gnificant changes to la nd use patterns. If, however, the demand for

canola and sunflower oil were to decline, growers have the option to grow non-oil producing crops such as corn and wheat on acreage currently dedicated to canola and

sunflower.

Growers make planting decisions for all cr ops based on market demand, commodity prices, and the agricultural needs and practices specific to their acres. Because of this, it is not anticipated th at the introduction of MON 87705 will result in a significant impact to canola and sunflower growers or to agri cultural land use in those geographic areas where canola and sunflower are currently grown.

5 Comparisons of canola and sunflower acreage established from databases at http://usda.mannlib.cornell.edu/usda/current/htrcp/Monsanto Company 09-SY-201U Page 448 of 471 Figure P-1. Harvested U.S. Acres of Canola, Sunflower and Soybean

Source: http://www.agcensus.usda.gov/Publications /2007/Online_Highlights/Ag_Atlas_Maps/Cr ops_and_Plants/index.aspMonsanto Company 09-SY-201U Page 449 of 471 P.D.2. Potential for Non-crop and Non-agricultural Impacts

Soybean ( Glycine max ) does not grow in the wild in the U.S. (Hymowitz and Singh, 1987; CFIA, 1996; OECD, 2000). Soybean does not grow and persist in unmanaged habitats and would not be exp ected to invade and/ or persist in the na tural environment, including streams, lakes, oceans or other a quatic environments. With the exception of suppression of the FATB and FAD2 RNAs resulting in an improved fatty acid profile, MON 87705 is similar to other commercial soybe an varieties currently grown in the U.S.

in that both MON 87705 and 92% of co mmercially grown soybean have the cp4 epsps gene that confers tolerance to the herbicid e glyphosate (USDA-NASS, 2008). Therefore, MON 87705 is expected to ha ve no significant impact to non-agricultural lands and aquatic systems beyond those related to co mmercially grown soybean. There are no changes in agricultu ral inputs needed to produce a crop from MON 87705 relative to commodity and specialty soybean and speci fically no altered pest or disease susceptibility. Hence, pesticide applicati ons on MON 87705 that may result in drift impacting non-crop or non-agricultural lands would be comparable to those used on conventional or currently av ailable soybean varieties.

Because MON 87705 produces the CP4 EPSPS pr otein conferring tolerance to the herbicide glyphosate it is antici pated that glyphosate will be used to control weeds in fields planted with MON 87705. Glyphosate has been ap proved for use by the U.S. Environmental Protection Agency and food and f eed tolerances have been established in the U.S. for its residues, since 1979. Glyphosate has a complete and comprehensive regulatory data base (toxicity, environmental fate, and ecological toxicity) that has been evaluated by EPA and it has been determined that “glyphosate will not pose unreasonable

risks or adverse effects to humans or the environment” (EPA, 2002) . More information on the impacts of glyphosate to animal and plant communities and threatened and endangered species may be found in Section P.D.7 of this appendix and Appendix O. Likewise a comprehensive discussion concer ning the development of glyphosate resistant weeds may be found in Appendix L.

P.D.3. Potential Impacts to Agricultural Practices

MON 87705 has been shown to be no differe nt from conventional soybean in its agronomic and ecological characteristics (see Se ctions VII, VIII, IX and X), and has the

same levels of resistance to insects and diseases as commercial soybean. A summary of agronomic practices for soybean production is presented in Sec tion IX of this petition. Because glyphosate-tolerant soybean is al ready grown on 92% of U.S. soybean acres (USDA-NASS, 2008), no significant impact is expected from the introduction of MON 87705 on current cultivation practices, including seeding, tillage, fertilizer and pesticide applications and crop rotation practices for soybean (Section IX). An analysis of expected impact to agricultural practi ces for commodity seed and certified seed production as well as potential impacts to specialty soybe an production are discussed

below.Monsanto Company 09-SY-201U Page 450 of 471 Potential Impact to Soybean Commodity and Specialty Soybean Production

Monsanto will seek regulatory approval fo r MON 87705 and its combinations, where required, with other biotechnology-derived so ybean varieties in all key soybean import countries with a functioning regulatory system to to support the fl ow of international trade. MON 87705 would provide food manufactu rers and consumers with a soybean oil having an improved fatty acid profile and greater thermal and oxidative stability for multiple uses. It would also provide growers with a higher value crop resulting in greater profit potential for U.S. soybean producers. Soybean growers and processors are accustomed to accommodating a diversity of sp ecialty soybean varieties in their normal handling of commodity soybean. Currently sp ecialty soybean varie ties are produced on approximately 12% of the U.S. soybean acreag e (see Section IX.B.2 ). Soybean growers have demonstrated an ability to provide cu stomers with their soyb ean product of choice. As discussed in Section IX, MON 87705 will be produced using IDP practices consistent with those used for other specialty soybean varieties. MON 87705 will be segregated from commodity soybean by producers as part of their contract and to maintain the value of the crop. Because soybean is primarily a self-pollinated crop with minimal gene movement and due to the identity preserva tion production system, no significant impact to commodity or other specialty soybean production is anticipated, should APHIS grant nonregulated status to MON 87705.

No cumulative impacts to commodity or othe r specialty soybean pr oduction are expected from deregulation. Soybean producers ha ve demonstrated thei r ongoing ability to produce multiple specialty soybean varieties in a diverse marketplace.

Potential Impact to Certified Seed Production

Certified seed productio n is a carefully managed process (Section IX.B.4). MON 87705 is not expected to impact certified seed production practices or production of other certified commodity or specialty soybean seed for reasons described in this section.

If MON 87705 is deregulated, seed productio n could occur within production systems already developed by seed producers for cer tified seed varieties (see Section X). MON 87705 has been thoroughly characterized and (with the excepti on of the modified fatty acid profile) is not agronomically or phenotypically different from currently commercial soybean. The implementation of ma nagement practices to avoid pollen from a biotechnology-derived crop in organic, specialty or conventional soybean seed or commodity seed production operations is dire ctly impacted by the nature of soybean pollination (Section IX.B.4). Soybean is a highly self-pollinated species that exhibits very low levels of outcrossing. Numerous studies on soybean cross-pollination have been conducted, and the published results, with and without supplemental pollinators, are

summarized in Table X-1. Under natura l conditions, cross-pol lination among adjacent plants in a row or among plan ts in adjacent rows ranged from 0 to 6.3%. In experiments where supplemental pollinators (usually bees) were added to the experimental area, crosspollination ranged from 0.5 to 7.74% in adjacent plants or adjacent rows. However, cross-pollination does not occu r at these levels over long distances. Cross-pollination

rates decrease to less than 1.5% beyond one meter from the pollen source, and rapidlyMonsanto Company 09-SY-201U Page 451 of 471 decrease with greater distances from the sour ce. The following cross-pollination rates at extended distances have been reported: 0.02% at 8.2 m of separation (Caviness, 1966), 0.05% at 5.4 m (Ray et al., 2003), and 0% at 6.5 m (Abud et al., 2003). Hence, certified soybean seed producers can and have effectiv ely implemented practices (e.g., isolation distances during the growing s eason, equipment cleaning duri ng harvest, and post-harvest separation of harvested seed) that allow them to maintain commercially acceptable levels of varietal purity.

No cumulative effects are anticipated to cer tified seed production from deregulation of MON 87705. The use of management practi ces that prevent trait movement and comingling of soybean varieties has resulted in production of soyb ean varieties with improved genetics over time. As a result of the implementation of these management practices, growers today can choose from nume rous varieties of s oybean including those used to produce commodity and sp ecialty soybean varieties.

Potential Impacts to Organic Soybean Production

Organic soybean markets typically enjoy a market premium offsetting the additional production and record-keeping costs associated with this production method (see Section IX.B.2). Organic farming operations as described by the National Organic Program, which is administered by USDA’s Agricultur al Marketing Service, requires organic production operations to have distinct, defined boundaries a nd buffer zones to prevent unintended contact with prohi bited substances or products of excluded methods from adjoining land that is not under an orga nic production management plan. Organic production operations must also develop and maintain an or ganic production system plan approved by an accredited certifying agent. This plan enables the production operation to achieve and document compliance with the Na tional Organic Standa rds, including the prohibition of the use of excluded methods. Excluded methods in clude a variety of methods used to genetically engineer or ganisms or influen ce their growth and development by means that are not possible und er natural conditions or processes. The use of biotechnology such as that used to produce MON 87705 is an excluded method under the National Organic Program [7 C.F.R. § 205.2].

Organic certification involves oversight by an accredited certifying ag ent of the materials and practices used to produce or handle an or ganic agricultural produc t. This oversight

includes an annual review of the certified operation’s organi c system plan and on-site inspections of the certified operation and its records. Although the National Organic Standards prohibit the use of excluded methods , they do not require testing of inputs or products for the presence of excluded methods. The presence of a detectable residue of a product of excluded methods al one does not necessarily cons titute a violation of the National Organic Standards. The unintentional presence of the products of excluded

methods will not affect the status of an or ganic product or operation when the operation

has not used excluded methods and has taken reasonable steps to a void contact with the products of excluded methods as detailed in an approved organic system plan. OrganicMonsanto Company 09-SY-201U Page 452 of 471 certification certifies that or ganic production and handling pr ocesses have been followed, not that the product itself is “free” from any particular substance.

Production systems designed prior to the intr oduction of MON 87705 or even prior to the introduction of biotechnology-derived soybean have allowed for production of soybean to

meet varied customer demands. In addition to the market segments that produce organic or conventional soybean, distinct identity-preserv ed specialty soybean with such traits as clear hilum or high protein have also been gr own and successfully marketed for specific food uses in domestic and export markets for many years (Cui et al ., 2004). The choice to grow biotechnology-deriv ed, organic or conventiona l soybean depends on many factors and the dynamics of the marketplace. The dynamics of the marketplace, choice

between various varie ties of soybean, and the existing pr oduction practices will not be impacted by the introduction of MON 87705.

Organic soybean producers utili ze production practices designed to specifically avoid the presence of both soybean products using co nventional herbicide or other pesticide treatments, as well as biotechnology-derived crops. These well esta blished practices to

avoid “excluded methods” will continue with the introduction of MON 87705 varieties. They include isolation zones, use of buffer rows surrounding the or ganic crop, adjusted planting dates and varietal selection (www.attra.ncat.org). The implementation of management practices to avoid pollen from a biotechnology-derived crop in organic or conventional soybean production operations is facilitated by the nature of soybean pollination. As noted previously in this petition, soybean is a highly self-pollinated species and exhibits very low level of out crossing. Hence, or ganic or conventional soybean producers can and have effectively implemented practices (e.g., isolation during the growing season, equipment cleaning durin g harvest, and post-harvest separation of harvested seed) that allow them to avoid the presence of biotechnology-derived soybean and maintain organic or c onventional production status.

Currently, biotechnology-derived herbicide tolerant soybean is planted on 92% of the soybean acreage (USDA-NASS, 2008) and th e Roundup Ready soybean system – that is, planting Roundup Ready soybean a nd applying glyphosate in crop – has become the most widely used weed control program in U.S. soybean production. Despite the high adoption rates of Roundup Ready soybean by gr owers, organic and conventional soybean production remains an option for farmers who choose to produce using these production practices or varieties of soybean. The d ecision to grow organic, conventional, or biotechnology-derived soybean varieties is typically an economic one based on market dynamics. Organic soybean producers and those growing convent ional soybean for sensitive non-biotechnology markets typically enjoy a market premium offsetting the additional production and record-keeping cost s. While the widespread adoption of Roundup Ready soybean has reduce d the number of conventional soybean varieties that are available, conventional and organically produced soybean seed is currently available from numerous seed suppliers (Table P-1). Additional information on organic seed sources is provided though the U.S. Agri cultural Marketing Service (AMS) at:Monsanto Company 09-SY-201U Page 453 of 471 www.ams.usda.gov. Thus, grower s have a choice in the soyb ean variety they plant, and this is not expected to change w ith the introduction of MON 87705.

Buyers recognize that where there are biotechnology-derive d crop varieties on the market, as with soybean, a guarantee that a commodity crop is 100% “free” of biotechnology-derived material is not feasible based on th e limitations of testing and sampling methodology (Born, 2005). While th e presence of a biotechnology-derived product like MON 87705 is unlikely in in stances where producers utilize production practices designed to avoid biotech presence, in some instances buyer allowances

between 0.1 to 5% biotechnol ogy-derived commodity soybeanin organic soybean are specified. This also is consistent with the USDA National Organi c Program allowing for detectable residues of excluded methods (i ncluding biotechnology-de rived crop products) as long as the producer has take n steps to avoi d those methods (www.ams.usda.gov/nop/Q&A.html). Similarly, international regulatory organizations have recognized that testing and sampling methodologies limit the ability to confirm that commodity or specialty soybean is 100% free of biotechnolo gy-derived material. Thus, they have set allowable tolerances for this material in conventional products to support food labeling and traceability laws. These tole rances allow from 0.9% (European Union) up to 5% (Japan) of the food to be biot echnology-derived in pr oducts considered

“conventional.” Levels above the threshol d may trigger special labeling. Thus, de

minimis levels of approved biotechnology-derived soybean mav be allo wable in certified organic or conventional soybean.

Table P-2. Organic and Conventional Soybean Seed Sources

Organic Soybean Seed Sources* : Conventional Soybean Seed Sources

Albert Lea Seed House AgVenture Seeds (modified oil)

Blue River Hybrids Campbell Seed (modified oil)

Golden Grains Becks Hybrids (food grade)

Great Harvest Organics Monsanto (Asgrow)

Greis Seed Farm Schillinger Seed

Lancaster Ag Products Pioneer

Lawler Farm Center Soy Genetics

Prairie Gold Seeds Stewart Seed (modified oil)

Superior Organic Grains, Ltd Stine Seed

Walter Seed and Honey Co Syngenta - multiple brands

Terral Seed
Various State Crop Improvement

Or g anizations

* From: www.organicgrains.ncsu.eduMonsanto Company 09-SY-201U Page 454 of 471 P.D.4. Potential Impacts to Raw or Pr ocessed Agricultural Commodities

Within this petition, extensive data have been presented relating to plant growth parameters, disease susceptibility, insect sus ceptibility, and forage and seed composition of MON 87705 compared to conventional soybean varieties. These data indicate that there are no biologically relevant diffe rences between MON 87705 and conventional soybean, except for the deliberate change in the fatty acid profile of the oil and its glyphosate tolerance. Biot echnology-derived soybean varieties like MON 87705 undergo a voluntary food and feed consultation proces s with the FDA prior to release on the market. Monsanto will complete this c onsultation process prior to a commercial

introduction of MON 87705.

From a grain quality standpoint (e.g., fore ign material, damaged grain, etc; see www.gipsa.usda.gov for soybean grain standards) , the soybean produced from MON 87705 varieties is expected to be of co mparable quality to soybean commercially produced in the U.S. for commodity markets ba sed on the data presented herein. The low saturated fat, high oleic acid oil produced from MON 87705 s oybean is suitable for use in any application for which commodity soybean oil is currently used, including products such as vegetable oils, salad dressings, margarine, etc.

As described elsewhere in this petition, M ON 87705 and the low satu rated fat, high oleic acid soybean oil produced from MON 87705 will be produced and processed utilizing an identity preservation (IDP) system to capture the food quality value of the oil. Because other low saturated fat, high oleic acid oils such as canola and sunflower are already present in the commodity stream and are rout inely handled by proce ssors using standard industry processing methods as described in Appendix K, the introduction of MON 87705 will require no new inventions or processes. Processors routinely perform analyses for oil quality and fatty acid compos ition that enable them to capture the value of specialty soybean oils, to determine the appropriate customers and uses for various soybean and other oils, and to create the approp riate and requested ble nds of such oils for particular uses.

Soybean is typically processed into two ma jor fractions, the oil and meal, and several minor fractions including leci thin and protein isolate. The MON 87705 oil fraction will have the intended low saturated fat, high oleic acid profile, and will be produced and utilized within an IDP system . With the exception of low level residual oil, the other processed fractions derived from MON 87705 are compositionall y equivalent to processed fractions from commodity soybean, an d will be utilized in a manner similar to commodity soybean processed fractions (see Se ction VII.C). The m eal, and other non-oil processed fractions are intended to be distri buted into the commodity stream and will be marketed and sold in the commodity ma rket. Because of their compositional equivalence, it is not anticipated that deregulation of MON 87705 will have a significant impact on these commodity s oybean processed fractions.

During the development of MON 87705, Mons anto has engaged in dialogue with relevant grain handlers, processors a nd food companies regarding the uses andMonsanto Company 09-SY-201U Page 455 of 471 applications of low saturated fat, high oleic soybean oil. This dialogue has contributed to the understanding of the processing and use of MON 87705 soybean and the resulting oil and other fractions, a nd to the market and trade assessme nts set forth in this petition. Before implementing an IDP system for MON 87705-derived varieties, Monsanto will have additional dialogue with relevant stakeholders in the value chain to discuss and understand the potential impacts on commod ity oils and processed soybean products from the IDP production, handling and use of MON 87705 soybean and its low saturated fat, high oleic soybean oil. Based on that dialogue and any resulting refinements to the market and trade assessments, Monsanto will take the following steps, where applicable and appropriate:

1. refine plans for production, handlin g and processing of MON 87705-derived soybean varieties and the low saturated fat, high oleic s oybean oil (the IDP system) to address valid concerns raised by the stakeholders

2. assess potential imp acts on processed soybean produc ts due to the presence of low saturated fat, high oleic soybean o il in commodity oil, and collaborate

with stakeholders to su pport the evaluations needed to assess when potential impacts might occur, what processe s may be necessary to mitigate any potential negative impacts, and what potential positive impacts may occur based on the improved oil profile

3. engage in industry outr each and education rega rding the MON 87705 soybean and low saturated fat, high oleic soyb ean oil IDP system, including product handling and use

4. make available prior to commercializ ation a detection method or testing regime that enables identity verificati on of MON 87705 for its intended use

Based on our market and trade assessments, because MON 87705 soybean is appropriate for all the same uses as commodity soybean, the only likely potenti al impact on raw or processed commodities in the event MON 87705-derived varieties reach the commodity

channel would be a reduction in saturated an d polyunsaturated fats in commodity oil.

This reduction in saturated a nd polyunsaturated fats would improve the functionality of the oil and not impact the suitability of the soybean oil for anticipated end uses. The only impact would be an improvement in the nu tritional profile of the oil relative to commodity soybean oil. Based on this analys is, there would be no significant impacts on raw or processed soybean commodities due to USDA-APHIS granting nonregulated

status to MON 87705.

Based on this assessment, Monsanto does not anticipate any significant cumulative impacts on either raw or processed soybean commodities resulting from the deregulation of MON 87705. Food ingred ient and food manuf acturers routinely process, manage,

handle and blend a wide variet y of specialty oils derived from oilseed crops (including other modified oils produced by conventionally bred varieties or pr eviously deregulated events) with no adverse or cumulative effect s on their manufacturing processes or on the quality of their products. Si milarly, food manufactures will integrate low saturated fat,Monsanto Company 09-SY-201U Page 456 of 471 high oleic soybean oil into their existing ha ndling and manufacturing process, and are expected to have the equivalent experience with low saturate d fat, high oleic soybean oil, resulting in no adverse or cumulative eff ects due to USDA-APHIS granting deregulated

status to MON 87705.

P.D.5. Potential Impacts on Commercial Use of Soybean

The decision to deregulate MON 87705 would allow for breeding of this product with conventional and previously deregulated biotechnology-derived s oybean varieties of diverse genetic backgrounds. These varieties will include commercial varieties with low

linolenic acid levels which can further enhan ce the oxidative stability of the soybean oil. In addition, MON 87705 will be combined us ing traditional breeding methods with other biotechnology-derived traits, including glyphosate toleran ce (MON 89788), to deliver the best agronomic platform to farmers. It is expected that breeders and certified seed producers would use MON 87705 to develop vari eties and to supply seed for planned commercial markets in the U.S. Monsanto anticipates that commercial use of MON 87705 will include export of soybean and soybean products, and has described import approval submission plans elsewhere in the petition. Monsanto’s stewardship program for this product is de scribed in Section IX.I.

There are no statistically significant diffe rences observed in yield between MON 87705 and its conventional control (Table VIII-4 ). However, MON 87705 represents an

additional trait to thos e commonly selected for by soybean breeders. Such traits, whether conventionally bred or biotec hnology-derived, have the potential to impact yield gains (Fehr, 2007). Monsanto and its breeding co mpany partners have aggressive breeding programs in place which utilize increased populat ion size and yield testing, and advanced molecular selection tools. This progr am will ensure that MON 87705 yields are continually increasing comparab le to conventional commodity soybean, thus providing a competitive commercial soybean al ternative to U.S. growers.

P.D.6. Potential Impacts to Human Health and Safety

Most human interactions with soybean occu r through agricultural pr oduction, industrial operations, or through consumption. Therefore, this health and safety discussion will focus on food and feed safety as well as sa fety of workers exposed to MON 87705. The low saturated fat, high oleic soybean oil derived from MON 87705 soybean oil is suitable for use in products such as vegetable oils, sa lad dressings, margarine, etc., or in any application for which commodity soybean oil is used.

Under the Federal Food, Drug, and Cosmetic Act (FFDCA), it is the responsibility of

food and feed manufacturers to en sure that the products they ma rket are safe and properly

labeled. Food and feed derived from MON 87705 must be in compliance with all applicable legal and regulato ry requirements. Biotechnolo gy-derived crops for food and feed use undergo a voluntary consultation proc ess with the FDA prio r to release onto the market. Although a voluntary process, Monsanto routinely completes a consultation withMonsanto Company 09-SY-201U Page 457 of 471 the FDA prior to placing a new biotechnology derived crop product on the market. A list of completed consultations on biotechnology-derived crop pr oducts is available at the

FDA website.

Human Health

MON 87705 is genetically modi fied to suppress endogenous FATB and FAD2 RNAs in the developing soybean seed, thereby improving the fatty aci d profile to contain lower saturated fat, higher oleic and lower linoleic fatty acid levels. Ther efore, the improved soybean oil from MON 87705 has enhanced heat and oxidative stability, and, due to the lower levels of saturated fats, a healthie r fatty acid profile relative to conventional soybean.

MON 87705 contains only those fatty acids th at are presently found in soybean oil. MON 87705 has a fatty acid profile that is comparable to commercial high oleic vegetable oils (e.g., high oleic canola, hi gh oleic safflower, hi gh oleic sunflower),

traditional oils such as olive oil that has a long-history of consumption in the diet, and

canola oil that obtained U. S. FDA GRAS status.

Extensive compositional analyses of forage and seed were conducted on samples from replicated, multi-site field trials to compare the composition of MON 87705 to a conventional soybean control and to commer cially available soybean varieties (see Section VII for details). The compositio nal analyses of MON 87705 confirmed the intended changes to four major soybean oil fatty acids. All other components analyzed in MON 87705 seed and forage were either not st atistically significantly different compared to a conventional control, or, if significantl y different, were within the 99% conventional soybean tolerance interval with the exception of eicosenoic acid. This value (0.34% FA)

fell outside the tolerance intervals. Howeve r, eicosenoic acid ha s a history of safe consumption at higher levels in other commonl y consumed vegetable oils such as canola oil (4.3% FA), peanut oil (1.7% FA), high ol eic safflower oil (0.5% FA) and high oleic sunflower oil (0.5% FA) (Codex 2005).

The Dietary Guidelines for Americans (U SHHS, 2005) recommend that saturated fat intake should be kept below 10% of tota l daily caloric intake and below 7% for

individuals with elevated lo w density lipoprotein (LDL) blo od cholesterol. The Dietary

Guidelines also recommend that most diet ary fat should come fr om PUFA and MUFA sources, such as vegetable oils. When MUFA s, such as oleic acid, are substituted for saturated fat in the diet, LDL cholesterol de creases. Likewise, diets high in PUFAs, such as the omega-6 fatty acid linoleic acid, are a ssociated with a favorab le blood lipid profile,

and diets that contai n 5-10% of energy as omega-6 PU FAs may confer benefits on coronary artery disease mortality.

MON 87705 soybean oil contains approximately 6% saturate d fatty acids, approximately 76% 18:1 oleic acid and approximately 10% 18:2 linoleic acids levels. The fatty acid profile of MON 87705 is similar to a variety of other widely consumed vegetable oilsMonsanto Company 09-SY-201U Page 458 of 471 such as canola and olive oils (Table P-3) for which the U.S. FDA has approved a qualified health claim related to coronary heart disease. 56 Fatty acid profiles similarly higher in oleic acid than conve ntional soybean oil are also found in other vegetable oils such as high oleic safflower and sunflower oils and in nut s such as filberts, almonds, pistachios and pecans (Gunstone, 1994). These edible fats and oils are available for consumption in a wide variety of products including shortenings, salad and cooking oils, margarine and mayonnaise.

Table P-3. Comparison of Fatty Acid Profiles from Plant Sources

Oil %* Saturated

Fat %* Oleic Acid (18:1) %* Linoleic Acid (18:2) %* Linolenic Acid (18:3) %* PUFAs

Canola 6 57 26 10 32

MON 87705

Soybean 6 76 10 7 17

Conventional Soybean 15 23 53 8 60

Olive 13 78 7 1 8

Palm 50 38 11 1 12

Coconut 92 6 2 0 2

Source: Padley et al., 1994.

*% of total fatty acids

A dietary assessment was conducted in wh ich MON 87705 replaced conventional liquid soybean oil in salad dressings, margarin e and spreads, mayonnaise and home use of liquid soybean oil. The assessment indicates that intake of the saturated fat (16:0 palmitic acid) would decrease from 14.4 g/day to 13.8 g/day representing a decrease of 3.8% on a mean per capita basis. A summary of this assessment can be found in Appendix M. A decrease in dietary saturated fat intake is consistent with current dietary recommendations (Lichtenstein et al., 2006; USHHS, 2005; WHO/FAO, 2003). There was an overall increase in the total daily in take of oleic acid from 27.3 g/day to 30.3 g/day representing an increase of 10.8% on a mean per capita basis. The oleic acid increase post-replacement is expected, since, similar to olive oil or canola oil, the oleic acid fraction in MON 87705 soybean oil is hi gher than the commodity soybean oil it replaced. Oleic acid is not an essential fatty acid and no recommended intake level has been set by the Institute of Medicine of the National Academies (IOM, 2002); however, as stated above, it is recognized that there is an inverse rela tionship between the intake of

MUFAs like oleic acid and the total cholesterol (TC) :HDL cholesterol (HDL-C)

concentration ratio.

56 http://www.cfsan.fda. gov/~dms/qhccanol.html, http://www.cfsan.fda.gov/~dms/qhcolive.htmlMonsanto Company 09-SY-201U Page 459 of 471 In summary, the nutritional impact from th e use of MON 87705 soyb ean oil in targeted

foods under the intended conditions of use, is estimated to re sult in changes in fatty acid consumption that are in line with current dietary guidelines for fatty acid intake. Therefore, MON 87705 is regarded to be as safe and nutritious as conventional soybean

for food use.

MON 87705 produces a lower satu rated fat, high oleic soyb ean oil that has improved oxidative stability and, due to the lower levels of saturated fats, a healthier fatty acid profile relative to comodity s oybean oil. As discussed prev iously, Monsanto will provide the U.S. FDA with information on identity, f unction, and characterization of the genes, including expression of the ge ne products. Additionally, information on the safety of the improved fatty acid profile in MON 87705 s oybean oil, including a dietary risk assessment, will be provided to the U.S. FD A for evaluation including a dietary risk assessment. On the basis of the assessment of laboratory data and scientific literature, it

is reasonable to conclude that MON 87705 is safe for food and feed use.

MON 87705 also expresses the CP4 EPSPS protein throughout the plant conferring

tolerance to glyphosate, which is the active ingr edient in the Roundup family of agricultural herbicid es. It is structurally homologous to EPSPS proteins that are part of the amino acid synthesis pathway of all plants (Devine et al., 1993). The safety of any protein(s) newly introduced into a biotechnology-derived crop needs to be assessed (Delaney et al., 2008; ILSI, 2004). The safety of CP4 EPSPS prot ein present in biotechnologyderived crops has been extens ively evaluated (Harrison et al., 1996). The U.S. EPA has also reviewed the safety of the CP4 E PSPS protein and has esta blished a tolerance exemption for the protein and th e genetic material ne cessary for its production in or on all raw agricultural commodities (40 CFR § 174.52 3). This exemption was based on a safety assessment that included rapid digestion in si mulated gastric fluids, lack of homology to known toxins and allergens, and lack of toxicity in an acute oral mouse gavage study. A history of safe use is supported by the lack of any documented reports of adverse effects

since the introduction of other Roundup Ready crops in 1996.

Roundup Ready soybean is planted on 92% of U.S. soybean acreage (USDA-NASS, 2008). Consequently glyphosate is the most widely used herbicide in soybean. The toxicology of glyphosate has b een extensively reviewed. A summary of food and feed tolerances, regulatory approvals with associat ed dietary exposure assessments and recent chronic and short-term aggregate risk as sessments for glyphosate may be found in Appendix O.

Worker Safety

In the agricultural production of soybean, gr owers may be exposed to pesticides during application of chemicals to crops. Pl anting Roundup Ready soybe an and applying glyphosate in crop has become the standard weed control program in U.S. soybean production. MON 87705 will share that R oundup Ready weed management system, so any adoption of MON 87705 by growers cu rrently planting Roundup Ready soybean would not significantly change the commercia l soybean agronomic practices, or use ofMonsanto Company 09-SY-201U Page 460 of 471 pesticides such as herbicid es, associated with soybean production (see Section IX). Worker safety issues related to the use of pesticides dur ing agricultural production of MON 87705 will remain the same. A comprehe nsive human safety evaluation and risk assessment concluded that glyphosate has low toxicity to mammals, is not a carcinogen, does not adversely affect reproduction and de velopment, and does not bioaccumulate in mammals (Williams et al., 2000). This petition demonstrates that, other than the improved fatty acid profile of the soybean oi l and tolerance to th e herbicide glyphosate, MON 87705 is agronomically and phenotypicall y equivalent to conventional soybean. Thus the cumulative impacts due to agricultu ral management and processing practices of

MON 87705 would be no differe nt than those due to ma nagement practices of commercial soybean varieties. Similarly, ther e are no changes to indus trial processing of MON 87705 relative to the processing of commercial soybean, including specialty soybean.

P.D.7. Potential Impacts to Plant, Anim al and Microbial Co mmunities Including Threatened or Endangered Species and Biodiversity

The following section addresses potential im pacts due to deregulation of MON 87705 to plant and animal communities, including soil or ganisms. An overview of the potential impact of glyphosate in the envir onment may be found in Appendix O.

In assessing the potential impact to plant and animal communities, the potential for gene movement and introgression from MON 87705 was evaluated because movement and establishment of the gene and trait to related species could have indi rect impacts to plant and animal communities that extend beyond the original recipient organism. Monsanto considered two primary issues: 1) the potential for gene fl ow and introgression, and 2) the potential impact of introgression. The genus Glycine has approximately nine species,

with G. max being placed in the subgenus Soja along with one other species, G. soja

. G.

max is sexually compatible with only G. soja and no other Glycine species. G. max is the only Glycine species located in the United States. Therefore, the probability of gene flow and introgression of MON 87705 into other species in the U.S. is essentially zero (Stewart et al., 2003); thus, the potential impact of introgression of MON 87707 to sexually compatible relatives on plant and animal communiti es is nonexistent if USDAAPHIS were to grant the petition for nonregul ated status. Additional discussion of the potential environmental impact due to gene movement may be found in Sections X.D.

Animals

Soybean production systems in agriculture ar e host to many animal species. Mammals and birds may seasonally consume grain, and invertebrates can feed on the plant during the entire growing season. Animals that f eed primarily on soybean are seed-feeding insects and rodents found in ag ricultural fields. Rodents, such as mice or squirrels, may seasonally feed exclusively on soybean seeds. Thus, these animals may have a diet containing significant amounts of soybean seed s. Deer may also browse in soybean fields on the forage and on seed left after harvest.Monsanto Company 09-SY-201U Page 461 of 471 Agronomic practices used to produce MON 87705 will be the same as those used to produce other glyphosate-tolerant soybean, in cluding Roundup Ready soybean, that were planted on 92% of U.S. soybean acreage in 2008 (USDA-NASS, 2008) so any switch by growers from their current glyphosate-tolerant soybean to MON 87705 would not result in significant impacts on w ildlife compared to current soybean production.

Potential impacts to animals w ould be primarily based on th e effects of the introduced protein CP4 EPSPS and the suppression of endogenous FATB and FAD2 RNAs resulting in the improved fatty acid profile of th e MON 87705 soybean oil. As discussed previously, there is no meaningful risk to animal or human health from dietary exposure to CP4 EPSPS or the improved fatty acid profile from MON 87705 soybean oil. There are no toxic properties associated w ith either the CP4 EPSPS protein or with the improved fatty acid profile produced by MON 8770 5. Furthermore, the composition of the seed, forage and meal produced by MON 87705 is unchanged from conventional soybean meal. This

information indicates that there would be no negative effects to mammals that forage on MON 87705. Similarly, it is expected that there would be no impact to birds or other animals that may consume soybean forage or soybean seed. During field trials no changes in insect feeding damage were observed (see Section VIII) indicating similar insect susceptibility for MON 87705 co mpared to conventional soybean. As

MON 87705 exhibits no toxic effects on animal s or pollinators of other plants in or around fields cultivated with MON 87705, they will not be affected.

Ninety-two percent of soybean acreage in the U.S. is herbicide-tolerant, with most of these acres planted with Roundup Ready s oybean (USDA-NASS, 2008). Consequently, glyphosate is the most widely used herbicid e in soybean production. A comprehensive human safety evaluation and risk assessment concluded that glyphosat e has low toxicity to mammals, is not a carcinogen, does not adversely aff ect reproduction and development, and does not bioaccumulate in mammals (Williams et al., 2000; Appendix O). An ecotoxicological risk assessment concluded that the use of glyphosate does not pose an unreasonable risk of adverse effects to non-target species, such as birds and fish, when used according to label directions, nor does it pose an unreasonable risk of adverse effects to insects outside of the applicati on area. On the basis of this analysis, deregulation of MON 87705 will not result in significant impacts on animals, including insects that live near or in soyb ean fields containing MON 87705.

Plants

Soybean production systems in agriculture are ho st to many plant species. Likewise, the environment surrounding a soybean field varies in plant composition depending on the region. In certain areas, soybean fields ma y be bordered by other soybean, corn or other crops; fields may also be surrounded by woode d and/or pasture/gras sland areas, as well as aquatic environments. Ther efore, the types of vegetati on, including w eeds, around a soybean field depend on the area where the soybean is planted. A va riety of weeds dwell in and around soybean fields; those species will also vary depending on the region where the soybean is planted.Monsanto Company 09-SY-201U Page 462 of 471 If MON 87705 is granted nonregul ated status, agricultural pr actices that are used for biotechnology-derived herbicid e-tolerant commercial soybean grown on the majority of U.S. acreage would be used for plant manage ment during the cultivation of MON 87705.

MON 87705 does not exhibit characteristics a ssociated with weedy growth and will not compete with plants found outsi de of agricultural production. Weeds within fields of MON 87705 will be managed using mechanical, cultural, and chemical control measures, as weeds are now managed in commercial soybean systems. Monsanto’s glyphosate label provides information regarding appropr iate conditions for a pplication of Roundup herbicide agricultural herbicides that ar e designed to minimize damage to adjacent vegetation. Therefore, the presence of the herbicide- tolerance trait in MON 87705 and the improved fatty acid profile trait is not expected to have a significant impact on surrounding plant communities.

Threatened and Endangered Species

No significant impact to threatened and endangered species is expected from the

introduction of MON 87705. Monsanto ha s considered the potential impact of MON 87705 on federally listed Threatened or Endangered Species (TES) and species proposed for listing, as provide d under Section 7 of the Endange red Species Act. In this analysis, Monsanto considered the biology of MON 87705, as well as typical agricultural practices associated with cultivation of s oybean. As previously noted, consumption of the CP4 EPSPS protein has no toxicity in la boratory testing with mice (see Section X.B.1.). MON 87705 does not express any additional proteins or natural toxicants that are known to directly or indire ctly affect a listed TES or species propos ed for listing by

the U.S. Fish and Wildlife Service. A dditionally, MON 87705 contains only those fatty acids that are presently found in soybean oil. MON 87705 has a fatty acid profile that is comparable to commercial high oleic vegeta ble oils (e.g., high ol eic canola, high oleic safflower, high oleic sunflower), that are currently grown inthe U.S.

MON 87705 is not sexually compatible with a federally listed TES or a species proposed for listing. The only TES animal listed that occupies habitat that is likely to include soybean fields and that might feed on soyb ean is the federally Endangered Delmarva Peninsula Fox Squirrel, ( Sciurus niger cinereus ), found in areas of the mid-Atlantic

Eastern seaboard 2 . It is known to utili ze certain agricultural lands readily, and its diet includes acorns, nuts/seeds of hickory, beech, walnut, and loblolly pine; buds and flowers of trees, fungi, insects, fruit, and an occasional bird egg. Given all these factors and the

lack of noted adverse effects of the CP4 EPSPS protein an d MON 87705 on mice and other non-target organisms, respectively, it is concluded that MON 87705 will not have an effect on the Delmarva Peninsula Fox Squirrel.

No impact to any threatened or endangered plant species is expected from the cultivation

of MON 87705. Like other G. max , MON 87705 will likely be a poor competitor with native vegetation, has no sexually-compatible relatives in the U.S. and will not survive

2 http://ecos.fws.gov/tess_public/SpeciesReport.do ; [Accessed May 14, 2009].Monsanto Company 09-SY-201U Page 463 of 471 outside of cultivation. Thus, there is no opportunity for MON 87705 to interbreed with any plant species or displace natu ral vegetation in the U.S.

In a TES risk assessment previously provi ded to USDA-APHIS in support of Roundup Ready alfalfa petition 04-110-01p Monsanto iden tified some plant but no animal species that may be at risk from the use of gl yphosate-based herbicides in the Roundup Ready crop system this assessment is summarized in Appendix O. To mitigate these potential risks, Monsanto developed Pre-Serve ( www.pre-serve.org), a web-based program designed to protect TES plant species fr om potential impacts resulting from the agricultural use of herbicides that contain glyphosate. Pr e-Serve instructs growers to observe specific precautions when sprayi ng glyphosate herbicides on Roundup Ready crops near TES plant species that may be at risk.

Soil Microorganism

No adverse effects on soil microorganisms are associated with MON 87705 nor do the

characteristics of the CP4 E PSPS protein or the improved fa tty acid profile pose any concern to soil microorganisms. Monsanto pr esented data in this petition (see Section VIII) demonstrating the lack of impact to symbiotic microbes associated with soybean plants. The B. japonicum -soybean symbiosis of MON 87705 was not changed as a result of the introduction of the CP4 EPSPS prot ein and the improved fatty acid profile compared to a conventional s oybean control. MON 87705 contai ns only those fatty acids that are presently found in soyb ean oil, and has a fatty acid pr ofile that is comparable to other commercial high oleic vegetable oils. The fatty acids found in MON 87705 have a long history of safe use in human and animal consumption, are naturally present in the environment, and will be br oken down and utilized by soil microorganisms in a manner similar to fatty acids found in other food crops.

On the basis of these observations and in conjunction with related phenotypic measurements for MON 87705, no impact on so il microorganisms or soil arthropods is expected from deregulation of MON 87705.

Biodiversity

Analysis of available information indicates that MON 87705 exhibits no traits that would

cause increased weedine ss, that its unconfined cultivati on would not lead to increased weediness of other sexually compatible rela tives (of which there are none in the United States), and it is unlikely to have effects on non-target organisms common to agricultural ecosystems or threatened or endangered spec ies recognized by the U.S. Fish and Wildlife Service. The use of herbicides in agricult ural fields is likely to indirectly impact biodiversity by decreasing weed species presen t in the field. Agricultural fields are purposefully managed to be weed-free resulting in great er economic benefit to the grower. Ninety-six percent of soybean acreag e in the U.S. was treated with glyphosate in 2006 (USDA-ERS, 2007). Therefore introduc tion of MON 87705 is unlikely to affect the animal or plant communities found in commercial soybean production systems due to both the lack of toxicity of either the CP4 EPSPS protei n or the improved fatty acid profile. Because MON 87705 will likely repl ace soybean varieties that already haveMonsanto Company 09-SY-201U Page 464 of 471 glyphosate tolerance, no significant impacts fr om its introduction are anticipated. For an overview of cumulative impacts from dere gulation of glyphosate-tolerant crops see Appendix N. Based on this analysis, it is concluded that deregulation of soybean varieities containing MON 87705 would have no significant impact on biodiversity.

No impacts to plant and animal communities or threatened and endangered species have

been identified for MON 87705. No cu mulative impacts to plant and animal communities or threatened and endangered species have been identified.

P.D.8. Other Cumulative Effects

Conventional Breeding with Other Biotechnology-derived or Conventional Soybean

Varieties

As previously mentioned, several biotechno logy-derived soybean crop varieties have been deregulated or are under consideration fo r deregulation, and a list of the events codes deregulated by USDA is presented in Table P-5 below. MON 87705 may be bred with these deregulated biot echnology-derived soybean crop pr oducts as well as with conventional soybean, creating new improved vari eties. APHIS has de termined that none of the biotechnology-derived individual soybean products it has deregulated display increased plant pest characteristics and that any progeny derived from crosses of these soybean crop products with other conven tional or biotechnology- derived soybean are unlikely to exhibit new pl ant pest properties.

An assessment of the stabil ity of the genetic insert in MON 87705 was conducted, and data have been presented in this petiti on demonstrating that MON 87705 is stable in progeny. Having established that the genetic material is stable and that MON 87705 is inherited in a Mendelian fashion, a nd based on experience with MON 87705 in Monsanto’s plant breeding program, it can be concluded that the phenotype of

MON 87705 is likewise stable. C onventional breeding has an es tablished history of safe use, and use of MON 87705 in breeding progra ms is expected to behave in a manner

similar to other conventional traits and biotech nology-derived traits. For example, in an assessment by McCann et al. ( 2005), it was shown that duri ng three years of breeding into multiple varieties, the composition of glyphosate-tolerant soybean remained equivalent to that of conventional soybean. Given that there have been no plant pest characteristics associated with MON 87705, or with any of the pr eviously deregulated events listed below, no significant impact s are expected to other soybean varieties through the use of MON 87705 in breeding program s and in combination with any of the previously deregulated soybean crop products.

All biotechnology-derived soybean products on the market today have satisfactorily completed the FDA consultation process estab lished to review the safety of foods and feeds derived from biotechnology-derived crops for human and animal consumption (see Table P-5). As mentioned a bove, breeding with modified oil soybean products on this list, while not currently envisioned, would pr edictably alter the fatty acid composition of MON 87705 (i.e., modify the oil profile to a combination of the individual traitMonsanto Company 09-SY-201U Page 465 of 471 characteristics). No impacts to public health (e.g., food or feed safety) are expected due to combination of these events through conve ntional breeding because the deregulated events have a history of safe use and on the basis of knowledge of the type of modifications made to each of the deregulat ed events, the biochemical pathways are not likely to unexpectedly interact or result in the production of novel constituents.

The decision to deregulate MON 87705 would also allow for breeding of this product with conventional soybean vari eties of diverse genetic back grounds. These varieties will

include commercial varieties with low linolenic acid levels which can further enhance the oxidative stability of the soybean oil. In addition, MON 87705 will be combined using traditional breeding methods with other biotechnology-derived traits, including glyphosate tolerant MON 89788. This comb ined trait product will incorporate the improved fatty acid profile of MON 87705 with the superior germplasm and 4-7% yield advantage of MON 89788 to deliver the best ag ronomic platform to farmers. No impacts to public health (e.g., food or feed safety) or environmental safety are expected due to the breeding of MON 87705 with these other soybean varieties because th ese varieties have an established history of safe use.

Furthermore, the process of conventiona l breeding to combine biotechnology-derived traits or biotechnology-derived and conven tional varieties to produce combined trait products would likely identify and rem ove off-types during development of new varieties. Breeders use standard testing a nd assessment procedures to further examine and confirm the equivalence of the combined trait products, compared to the single event products, in terms of phenotypes, agronomic charact eristics, and the effica cy of the traits.

Table P-5. Deregulated or Submitted Bi otechnology-derived Soybean Products

Phenotype ID Code(s) Inst itution Date Deregulated

High Oleic Acid DP-3Ø5423-1 Pioneer Submitted

Glyphosate Tolerant MON 89788 Monsanto February, 2007

Phosphinothricin

Tolerant GU262 AgrEvo October, 1998

Phosphinothricin

Tolerant A5547-127 AgrEvo May, 1998

Altered Oil Profile G94-1, G94-19, G-168 DuPont May, 1997

Phosphinothricin

Tolerant W62, W98, A2704-12, A2704-21, A5547-35 AgrEvo August, 1996

Glyphosate tolerant 40-3- 2 Monsanto May, 1994

Source: http://www.aphis.us da.gov/brs/not_reg.html

Economic Impacts of Biot echnology-derived Products

In a recent study, economist s Brookes and Barfoot (2006) quantified the cumulative economic and environmental impacts of bi otechnology-derived crops grown during the past eleven years (1996-2006). The authors report that biotechnol ogy-derived crops haveMonsanto Company 09-SY-201U Page 466 of 471 resulted in substantial globa l economic and environmental benefits. In the U.S. the impact of herbicide-tolerant soybean has primarily been to reduce the cost of production. In the early years of adoption the savings we re between $25-34/ha. In more recent years the cost savings have risen to $60-78/ha ba sed on comparison of he rbicide regimes that would be required to deliver a co mparable level of weed control. It is estimated that the cumulative U.S. farm income benefit from 1996-2006 was approximately $8.73 billion USD. Other soybean production countries (Arg entina, Brazil, Canada, South Africa) have also experienced cost savi ngs from the adoption of bi otechnology-derived herbicidetolerant soybean varieties. In addition to cost savings , countries like Romania and Mexico have also experienced yield ga ins (31% and 9%, respectively) from improvements in weed control.

It is not known how many U.S. acres MON 87705 will be grown on when commercialized. However, MON 87705 may provi de benefits to U.S. soybean producers

similar to herbicide- tolerant biotechnology-derived s oybean commercialized on broad acres across the U.S., and provide growers with another high-value speci alty soybean product option.

P.E. Highly Uncertain, Unique or Unknown Risks

MON 87705 has been thoroughly characterized and data submitted in the petition demonstrate that it poses no increased plant pe st risk compared to conventional soybean. USDA-APHIS has previously deregulated th irteen biotechnology-derived soybean crop products. MON 87705 offers a low saturated fat, high oleic soybean oi l alternative that has significantly improved heat and oxidativ e stability. Introduction of previous biotechnology-derived crops have resulted in no unexpected effects on the quality of the

human environment as define d under NEPA and have provi ded benefits to growers,

consumers and the environment. In this respect, a decision to deregulate a new biotechnology-derived soybean product is not precedent setting nor are the effects to the quality of the human environment hi ghly uncertain or unpredictable.

P.F. Summary

MON 87705 has been thoroughly characterized and the extensive body of information presented in Sections I through X of this petition demonstrates that MON 87705 does not present a plant pest risk, has no significant impact on threaten ed or endangered species or biodiversity, and will not imp act the commercial interests of soybean producers or those involved in the marketing and sale of soybean and soybean products. The introduction and adoption of specialty soybean crop products have benefited farm income in the U.S. The amount of land devoted to farming (specifi cally to corn or s oybean) has not changed

with the introduction of biotechno logy-derived crops. Similarly, no significan t change in the use of agricultural land or amount of land devoted to farming wo uld be expected to

occur with the commercial introduc tion of MON 87705. MON 87705 will utilize common cultivation practices typically empl oyed for production of commodity soybean and many types of specialty soybean, and ma nagement practices typically used forMonsanto Company 09-SY-201U Page 467 of 471 specialty soybean (i.e., IDP pr oduction practices). Hence, agricultural practices would not be impacted if MON 87705 were deregulated.

The opportunity for growers to produce soybean with a low saturated fat, high oleic acid fatty acid profile that has significantly improve d heat and oxidative stability relative to conventional soybean is a positive benef it from an economic and human health perspective. Additionally, MON 87705 soybean and its fractions have, other than the intended changes to fatty acid composition, been demonstrated to be equivalent to conventional soybean. Therefor e, MON 87705 soybean and its fractions are as safe and as wholesome for food and feed purposes as conventional soybean. For these reasons, the proposed action to gran t nonregulated status to MON 87705 does not represent a significant impact to the environment.Monsanto Company 09-SY-201U Page 468 of 471 Appendix P References

Alt, J.L., W.R. Fehr, G.A. Welke, and J.G. Shannon. 2005. Transgressive segregation for oleate content in three soybean popul ations. Crop Science 45:2005-2007.

Born, H. 2005. Marketing organic grai ns. http://www.attra. ncat.org/attrapub/PDF/marketingorganicgrains.pdf [Accessed May 19, 2009].

Brookes, G and P. Barfoot. 2006. Global imp act of biotech crops: socio economic and

environmental effects 1996-2006. PG Economics Ltd., UK.

CFIA. 1996. The biology of Glycine max (L .) merr. (soybean). Biology Document BIO1996-10, O. Canadian Food Inspection Agency, Ontario

Codex. 2005. Codex Standard for named ve getable oils. Codex Alimentarus.

Cui, Z., A.T. James, S. Miyazaki, R.F. Wils on, and T.E. Carter Jr. 2004. The breeding of specialty soybeans for tradit ional and new soyfoods. Pp 264-322 in Proceedings of the American Oil Chemists' Society.

Delaney, B., J.D. Astwood, H. Cunny, R.E. Conn, C. Herouet-Guicheney, S. Macintosh, L.S. Meyer, L. Privalle, Y. Gao, J. Mattss on, and M. Levine. 2008. Evaluation of protein safety in the context of agricultural bi otechnology. Food and Chemical Toxicology. 46 Suppl 2:S71-97.

Devine. M.D., S.O. Duke and C. Fedtke. 1993. Inhibition of Amino Acid Biosynthesis. Pages 251-294 in Physiology of Herbicide Action. PTR Prentice-Hall, Inc., Englewood Cliffs, New Jersey.

Dutton, H.J. 1963. Kinetics of linolenate hydr ogenation. Journal of the American Oil Chemists’ Society 40:35-39.

Dutton, H.J., C.R. Lancaster, C.D. Evans, a nd J.C. Cowan. 1951. The flavor problem of soybean oil. VIII. Linolenic Acid. Jour nal of the American Oil Chemists’ Society

28:115-118.

EPA. 2002. Biopesticides regi stration action document ( Bacillus thuringiensis Cry2Ab2 protein and its genetic material necessa ry for its production in cotton). U.S. Environmental Protection Agency Chemical PC Code 006487

.

FDA. 2006. Food Labeling: Trans Fatty Acids in Nutrition Labeling, Nutrient Content Claims, and Health Claims.Monsanto Company 09-SY-201U Page 469 of 471 Fehr, W.R. 2007. Breeding for modified fatty acid composition in Soybean. Crop Science 47(S3):S72–S87

Graef, G., B.J. LaVallee, P. Tenopir, M. Tat, B. Schweiger, A.J. Kinney, J.H.V. Gerpen, and T.E. Clemente. 2009. A high-oleic-acid and low-palmitic-acid soybean: Agronomic performance and evaluation as a feedstock for biodiesel. Plant Biotechnology Journal

7:411-421.

Gunstone, F.D. 1994. Fatty acid structure. Pages 1-20 in The Lipid Handbook. 2nd ed. F.D. Gunstone, J.L. Harwood and F.B. Padley (eds.). Chapman & Hall, Cambridge, Great

Btitain.

Harrison, L.A., M.R. Bailey, M.W. Naylor, J.E. Ream, B.G. Hamm ond, D.L. Nida, B.L. Burnette, T.E. Nickson, T.A. Mitsky, M.L. Ta ylor, R.L. Fuchs, and S.R. Padgette. 1996. The expressed protein in glyphosate-tole rant soybean, 5-enolpyruvylshikimate-3- phophate synthase from Agrobacter ium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice. Journal of Nutrition 126: 728-740.

Hu, F.B., M.J. Stampfer, J.E. Manson, E. Rimm, G.A. Colditz, B.A. Rosner, C.H. Hennekens, and W.C. Willett. 1997. Dietary fat intake and the risk of coronary heart disease in women. The New England Jour nal of Medicine 33 7(21):1491- 1492.

Hymowitz, T., and R.J. Singh. 1987. Taxonom y and Speciation. Soybean Monograph, Soybeans: Improvement, Production and Uses:23-48

ILSI. 2004. Nutritional and Safety Assessm ents of Foods and Feeds Nutritionally Improved through Biotechnology. http ://www.ift.org/cms/?pid=1000362.

IOM. 2002. Dietary fats: Total fats and fatty acids. Pages 8-57 and 422-541 in Dietary Reference Intakes for Energy, Carbohydrate, Fibe r, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. Panel on Macronutrients, . Institute of Medicine. National Academies Press, Washington D.C.

James, C. 2007. Global status of commerci alized biotech/GM crops : 2007. International Service for the Acquisition of Agri-biotech Applications. Cornell University, Ithaca,

New York.

Judd, J.T., B.A. Clevidence, R.A. Muesing, J.Wittes, M.E. Sunkin, and J.J. Podczasy. 1994. Dietary trans fatty acids: Effects on pl asma lipids and lipoproteins of healthy men

and women. American Journal of Clinical Nutrition 59:861-868.

Knothe, G. 2005. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Pro cessing Technology 86:1059-1070Monsanto Company 09-SY-201U Page 470 of 471 Lichtenstein, A.H., L.J. Appel, M. Brands, M. Carnethon, S. Daniels, H.A. Franch, B. Franklin, P. Kris-Etherton, W.S. Harris, B. Ho ward, N. Karanja, M. Lefevre, L. Rudel, F. Sacks, L. Van Horn, M. Winston, and J. Wylie-Rosett. 2006. Diet and lifestyle

recommendations revision 2006: a scientific statement from the American Heart

Association Nutrition Committee. Circulation 114:82-96.

McCann, M.C., K. Liu, W.A. Trujillo a nd R.C. Dobert. 2005. Glyphosate-tolerant soybeans remain compositionally equivalent to conventional soybeans (Glycine max. L.) during three years of field te sting. Journal of Agricult ure and Food Chemistry 53:5331-

5335.

Mensink, R.P. and M.B. Katan. 990. Effect of dietary trans fatty acids on high-density and low-density lipoprotein cholesterol levels in healthy subjects. New England Journal of Medicine 232(7):439-445

OECD. 2000. Consensus document on the biolo gy of Glycine max (L.) merr. (soybean). OECD ENV/JM/MONO(2000)9. Organiza tion for Economic Co-operation and Development, Paris.

Okkerse, C., A. de Jonge, J.W.E. Coenen, and A. Rozendall. 1967. Selective hydrogenation of soybean oil in the presence of copper catalysts. Journal of the American Oil Chemists’ Society 44:152-156.

Padley, F.B., F.D. Gunstone and J.L. Harw ood. 1994. Occurrence and characteristics of oils and fats. Pages 47-224 in The Lipid Handbook. 2nd ed. F.D. Gunstone, J.L. Harwood and F.B. Padley (eds.). Chapman & Hall, Cambridge, Great Btitain.

Ray, J.D., T.C. Kilen, A.C. Abel, and R.L. Paris. 2003. Soybean natural cross-pollination

rates under field conditions. Environm ental Biosafety Research 2:133-138.

Sankula, S. 2006. Quantification of the impacts on US agriculture of biotechnologyderived crops planted in 2005. National Ce nter for Food and Agriculture Policy, Washington, D.C.

Soyatech. 2008. Statistics. Soyatech, Manitoba, Canada.

Stewart, C.N., M.D. Halthill, and S.I. Warwick. 2003. Transgene introgression from genetically modified crops to their wild relatives. Nature Reviews 4:E1-E17.

USDA-ERS. 2007. Adoption of gene tically engineered crops in the U.S. U.S. Department of Agriculture, Washington, D.C

USDA-NASS. 2009a. Crop Production 2008 Summary. U.S. Department of Agriculture, Washington, D.CMonsanto Company 09-SY-201U Page 471 of 471 USDA-NASS. 2008. Acreage. U.S. Departme nt of Agriculture, National Agricultural Statistics Service. Washington, D.C.

USDA-NASS. 2006. Crop production 2005 summar y. U.S. Department of Agriculture National Agricultural Statistics Service.

USHHS. 2005. Dietary Guidelines for Am ericans. HHS Publication No: HHS-ODPHP2005-01-DGA-A. U.S. Department of Health and Human Services, Washington, D.C. www.health.gov/dietaryguideli nes [Accessed June 7, 2009].

WHO. 2003. Diet, Nutrition a nd the Prevention of Chronic Di seases. Report of a joint WHO/FAO Expert consultation. WHO Technical Report Series 915:55-56.

Williams, G.M., R. Kroes, and I.C. Munro. 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingr edient, Glyphosate, for humans. Regulatory Toxicology and Pharmacology 32:117-165.

Zock, P.L. and M.B. Katan. 1992. Hydrogenation alternatives: effects of trans fatty acids

and stearic acid versus linoleic acid onserum lipids and lipoproteins in humans. Journal of Lipid Research 33:399-410.CBI Deleted Copy

Page 1 of 127 

## Petition for the Determination of Nonregulated Status for Improved Fatty Acid Profile MON 87705 Soybean

ADDENDENDUM I

Market Impact of MON 87705 High Oleic Soybean Oil

Submitted April 7, 2011

OECD Unique Identifier: MON-87705-6

Monsanto Petition Number: 09-SY-201UCBI Deleted Copy

Page 2 of 127 

### Introduction

Overall total use of oils derived from plant sources (vegetable oil) for food and industrial
applications exceeded 14 million tons in North America in 2009. Due to its abundance, reliable
supply and price, soybean oil is on e of the most highly used of th e vegetable oils (Attachment I,
Table 3.1, pg 62). In 2009, soybean oil accounted for approximately 8.2 million tons of food and
1.2 million ton of industrial vegetable oil uses (S ee Attachment I, Part 1b, pg 17-18). Soybean
oil use in foods peaked at roughly 9.3 million tons in the 2006/2007 timeframe just before the
U.S. Food and Drug Administration required food labels to report trans-fat content in 2006.

( http://www.fda.gov/Food/LabelingN utrition/ConsumerInformation ). The demand for soybean
oil for frying and baking applications has declin ed in recent years due to the desire to lower
unhealthy trans-fat in the diet and changes in food labeling policy.

High oleic soybean oil produced using MON 87705 has a fatty acid profile that has reduced
saturated fats, and higher monounsaturated fat. Th ese properties, particularly the reduction in
polyunsaturated fats (18:2), resu lt in increased oil oxidative stability greatly improving oil
performance in frying and baking ap plications. The decrease in sa turated fat (16:0, 18:0) further
improves the nutritional profile of the oil for use in food preparation. MON 87705 will be bred

into Vistive ® low-linolenic soybean further improving the oil profile by lowering the level of
linolenic acid and eliminating th e need to hydrogenate the oil.

Monsanto has conducted a market and trade asse ssment to evaluate the potential impacts on
commodity soybean oil and other ve getable oils due to the introducti on of high oleic soybean oil.
The assessment considered that other vegetabl e oils including high olei c vegetable oils are
currently on the market and are routinely handled by oil processo rs and food formulators. The
system responsible for production, harvest, pr ocessing and handling of soybean and other
vegetable oils includes critical control points as well as economic incentives for identity
preserved soybean oils and other vegetable oils . Typically, the processor will assess the fatty
acid profile of the oil sold to a food manufacturer prior to sale. If the fatty acid profile were
different from commodity soybean oil, the oil co uld be blended (a common industry practice) to
reach the appropriate fatty acid c ontent based on specific food applications. On the basis of this

assessment it is concluded that the introduction of high oleic soybean oil w ill have little if any
potential for negative market impacts. In the event that comingling did occur, the economic
impact of comingling to a food manufacturer would be minimal and remedied through blending.

A summary of the intended uses for high olei c soybean oil, current processes for handling
vegetable oils and potential impacts to commodity soybean oil as well as other vegetable oils is
provided in this document.

® Vistive is a registered trademark of Monsanto Technology LLCCBI Deleted Copy

Page 3 of 127 

### Intended Uses of High Oleic Soybean Oil

Commodity soybean oil does not have the optimum fatty acid profile to satisfy many of the
needs of the food industry 1 . The fatty acid profile for soyb ean oil does not provide optimal
oxidative stability compared to other vegeta ble oils unless modified via hydrogenation or
through blending with othe r vegetable oils. Hydr ogenation creates unhealthy trans-fat making
soybean oil less desirable for use in some app lications. In addition, due to low oxidative
stability, commodity soybean oil is not highly stable for cooking and frying when used in these
applications, causing several operational challenges such as more frequent exchange of vegetable
oil stocks and/or replacement of oil and polymer ization, leading to fre quent costly equipment
cleaning. For these reasons, in rece nt years, soybean oil has lost sh are to other vegetable oils in
food applications (Wilson, 2004; Attachment I, Part 2, pg 38).

Given recent food labeling policy changes and m odifications to dietary guidelines, the food
industry is seeking vegetable oils that not only have enhanced f unctionality but minimize levels
of trans- and saturated fat; the balance of fatty acids (high oleic, low saturated fat, low linolenic)
is often difficult to achieve with current vegetabl e oil options. Soybean o il competes with other
oils such as palm, canola, sunflower, peanut and corn. These oils are generally blended to satisfy
food companies seeking to address functional or nutrition related needs. High oleic soybean oil
can provide a complementary option either as a straight replacem ent of blended oils or as a
blending alternative to end users. While ble nding achieves the food company’s objective, it is
costly due to increased handling, average oil price and required technical support (Attachment I,
Part 4, pg 75).

Through conventional plant breeding, MON 87705 will be bred with Vistive low-linolenic
soybean. Vistive low-linolenic soybean was de veloped through conventio nal plant breeding to
produce lower levels of linolenic acid allo wing food companies to avoid hydrogenation of
soybean oil helping to eliminate unhealthy trans fatty acids from the diet. The low linolenic
soybean has a mutation in the Fad3-1c and Fad3-1b genes that result in reduced levels of
linolenic acid. The Vistive low-linolenic soybean oil is currently commercially sold in the
United States. The soybean oil from MON 87705 in the Vistive low-linolenic soybean
background has the following specification: palmitic acid (< 4.0%); steari c acid (< 4.0%); oleic

acid (55 - 85%); linoleic acid (8 – 30%); and linolenic acid (< 4.0 %). The modification of the
fatty acid profile provides an equal or better alte rnative to vegetable oil options available today
and offers the opportunity to reduce the practice of blending which has become the solution for
fry applications to avoid trans-fats . The observed fatty acid profile of MON 87705 soybean and
MON 87705 in a Vistive low-linolenic soybean genetic background are presented in Table 1.

1 http://qualisoy.comCBI Deleted Copy

Page 4 of 127 Figure 1. Fatty acid synthesis in MON 87705 in a Vistive low-linolenic genetic background

Table 1. Fatty acid composition (% weig ht of soybeans) of MON 87705 soybeans and MON 87705 in a Vistive low-linolenic soybean background

Year C16:0 C18:0 C18:1 C18:2 C18:3

MON 87705

2007 1 2.36 3.31 76.47 10.10 6.69

MON 87705 in a Vistive low-lin olenic soybean background

2007 2 2.60 3.05 77.27 13.77 2.63

2008 2 2.47 2.97 75.30 15.60 2.96

2009 3 2.31 3.29 71.21 19.03 3.20

2010 4 2.20 3.43 76.83 13.73 2.47

1 Samples were obtained from field trials conducted in Chile in the 2007/2008 growing season. See USDA Petition

Number 09-201-01p.

2 Grown in Jerseyville, IL.

3 Samples were obtained from 8 U.S. locations (2 sites each in the states of IA, IL, IN, and OH). Value is mean of

the 8 individual site values.

4 Samples were obtained from 3 U.S. locations (2 sites in IL and one site in OH). Value is mean of the 3 individual

site values.

The safety of MON 87705 soybeans in a Vistiv e low-linolenic soybean genetic background is
based on the long-standing history of safe consum ption of the levels and types of fatty acids
contained in commercial vegetable oils with a fatty acid profile simila r to MON 87705 soybeansCBI Deleted Copy

Page 5 of 127 in Vistive low-linolenic b ackground. Monsanto has completed a safety assessment 2 of high oleic
soybean oil and high oleic soybean oil is gene rally recognized, among qualified experts, as
having been adequately shown to be safe under the conditions of its intended use. MON 87705
soybean in a Vistive low-linolenic soybean ge netic background does not contain any new fatty
acids that are not presently found in conventional food oils and the fatty acid prof ile of this oil is
similar to many other commercial oils currently available including canola, olive oil, high oleic
safflower oil, and high oleic sunflower oil (S ee Table 2). The oil from MON 87705 soybeans in
a Vistive low linolenic soybean genetic background provides options for food formulation that is
virtually trans fat-free, and, unlike palm oil, is low in saturated fat.

Table 2. Fatty acid composition (% weig ht) of MON 87705 in Vistive background in
comparison to soybean oil and other high oleic vegetable oils 1

Oil Saturated

fats trans fats Oleic acid Linoleic

acid Linolenic

acid Other fatty

acids

Soybean oil, all purpose 15.2 0.7 22.6 50.1 6.5 4.9

PH Soybean 2 24.7 34.1 31.4 4.5 0.2 5.1

MON 87705 in Vistive background 3 6.8 0.22 71.7 16.9 2.9 1.5

HOSO (305423) 4 14.27 0 70.6 5.5 7.2 3.4

Olive oil 13.8 0 71.3 9.8 0.8 4.3

HOLLCO 5 3 0 84 7 2 4

High oleic canola 6.5 0.8 70 14.3 2.6 5.8

Canola 7.6 1.6 60.6 17.7 6.4 6.1

High oleic Sunflower 9.7 0 82.6 3.6 0.2 3.9

1 The fatty acid profiles of PH soybean oil (partially hydrogenated soybean oil), olive oil, canola oil, high oleic canola oil, a nd

high oleic sunflower oil were obtained from the USDA nutrien t database (USDA 2008, Nationa l Nutrient Database for

Standard Reference. U.S. Department of Agriculture (USDA), Agricultura l Research Service (ARS), Nutrient Data Laboratory; Beltsville, Maryland. Available from : http://www.nal.usda.gov/fn ic/foodcomp/search/).

2 PH Soybean: partially hydrogenated soybean oil.

3 MON 87705 in Vistive background fatty acid composition is based on the pooled analysis of 8 oil lots.

4 HOSO (305423): high oleic soybean oil from Pi oneer/DuPont, defined in Delaney (2008).

5 HOLLCO: High oleic low linolenic ca nola oil, values from Möllers (2004).

2 See FDA GRAS assessment at: http://www.fda.gov/Food/GuidanceComplianceRegulatoryI nformation/GuidanceDocumen ts/FoodIngredientsandPac kaging/ucm061846.htm#Q1CBI Deleted Copy

Page 6 of 127 Vegetable oils are used in twelve broadly groupe d end use sectors: snacks, salad oils (use in
mayonnaise, salad dressings and other food products ), margarine, bottled oils (home use), par
fry, ingredient, confectionary, baked goods, industrial fry, restau rant, institutional, and food
service (Attachment I, Part 2, pg 37). High oleic s oybean oil is targeted fo r ten of the twelve end

use sectors with bottled and sala d oils not considered a commercia l target for high oleic soybean
oil because commodity soybean oil currently serving thes e sectors does not require
hydrogenation and products currently have acceptable shelf lives.

Industrial uses such as bio lubr icants and biodiesel are also possible end uses of high oleic
soybean oil. High oleic soybean o il is a renewable resource and is an attractive option as the
price of crude petroleum continues to increase. The increased leve ls of oleic acid in high oleic
soybean oil enhance the stability and lubricity of the oil making it a su perior bio lubricant
compared to petroleum based oils and a good f eedstock for biodiesel (Graef et. al, 2009).

Food and industrial end use sectors are currentl y supplied by commodity soybean oil as well as
domestic and imported blends of other vegetabl e oils (e.g. palm oil). Thus, high oleic soybean
oil would potentially replace commodity soybean oil and oil blends currently used for these
applications.



### Market Potential of High Oleic Soybean Oil

In order to assess the market potential for high oleic soybean oil, Monsanto commissioned a
study that included an in-depth an alysis of the vegetable oil market in North America that was
performed by LMC International (Attachment I). The study was conducted to assess the
potential demand for vegetable oil with the fatty acid properties of hi gh oleic soybean oil.

Factors considered included curr ent uses of vegetable oils and their physical properties, health
attributes of the oil, availability and price. The market potential estimate assumed that high oleic
soybean oil was used in the target food sector s described above as a direct replacement for
hydrogenated soybean oil and other vegetable oils (such as canola, corn, palm and sunflower)
used in food applications (Attachment I, part 5; pg 88). The demand for high oleic soybean oil is
price sensitive relative to other vegetable oils. To estimate of the maximum high oleic soybean the lowest price premium ($50/metric ton) was us ed. At the lowest price premium, high oleic
soybean would achieve a demand for the oil estimate d at 3.5 million metric tons. On the basis of
this analysis, approximately 16 million acres of high oleic soybean would satisfy the demand if
high oleic soybean oil were to replace current uses of hydrogenated soyb ean and other high oleic
vegetable oils in food and industrial applications (Att achment I, Part 5, pg 90) 3 . Additional acres
would likely be planted to supply high olei c soybean oil for indus trial uses. Based on
assumptions regarding product adoption and accep tance by the industry, Monsanto estimates it

would take at least a decade for high oleic soybeans to achieve this acreage and at peak

3 One bushel of soybean = 60 lbs and contains 10.7 lb oil (Wilson, 2004); average yield of soybean = 43.7 bushels per acre in 2010 (USDA NASS : http://www.nass.usda.gov)CBI Deleted Copy

Page 7 of 127 penetration, this level would represent approximately 23% of total planted soybean acres 4

. Subsequently, there would continue to be ade quate supply of both commodity and high oleic
soybeans to serve the vege table oil market needs.



### Production and Handling of Vegetable Oils

Crop production and distribution systems as well as oil processors and food manufactures have
demonstrated the ability to handle multiple vegetable oils derived from numerous crop species. A
summary of the current practices including critical cont rol points for production of soybean oil
and specialty soybean oil (e.g. Vistive low linolen ic) is provided below. There are numerous
vegetable oils on the market today including: palm, coconut, palm kernel, cottonseed, soybean,
groundnut, rapeseed and sunflower (Attachment I, Part 2, pg 38). Other minor use oils include
olive, corn, high oleic canola, high oleic sunflower oil and low linoleic soybean oil (Vistive).
The processing industry (e.g. Cargill, Bunge, ADM, etc.) and end users of such oils are highly
adept at handling these vegetable oils in their norma l course of business. As a result, processors
have developed methods to segregate multiple vege table oil feedstocks as they are financially
motivated to prevent mishaps with their cust omers. High oleic soybean, for planting and
subsequent processing, would be introduced to the market place as an option for processors to
supply food companies and those who seek a U.S. based vegetable oil option.

Once key global market approvals are obtained, oil from MON 87705 will be available as an
option for use by food processors and will be prod uced in an identity preserved (IDP) fashion
similar to the system that has been successf ully implemented for production of Vistive low
linolenic soybean and other specialty vegetable oils. IDP practices are implementd for value
added specialty soybean to captu re the enhanced value of the product and ensure that the enduser or processor receives the soybean with the desired identity, fatty acid profile and quality.
Vistive low-linolenic soybean was made availa ble to growers in 2005 and has been grown on

over 3.0 million acres since its initial launch.

A summary of the quality control systems asso ciated with seed production, crop production,
processing and end uses of high oleic s oybean oil is presented in Figure 2.

4 Assumes 70 million acres of soybean are planted.CBI Deleted Copy

Page 8 of 127 Figure 2. Production and use check points for high oleic soybean

Seed Production and Processing . Monsanto is a leader in crop biotechnology having
successfully introduced numerous biotech crops to the marketplace. Monsanto has developed
and implemented seed quality practices to as sure that soybean seed meets the standards
established for purity of a trait. These standards apply to all soybean seed sold by Monsanto and
are based upon measures that s eed producers put in place to a ssure the genetic purity of
improved planting seed. This system is used to assure that farmers receive seed of known
quality with a minimum level of off types.

The first step in the process for production of high oleic soybean is the production, processing
and delivery of high quality seed to the grower . The entire seed production process at the
majority of the seed companies and tollers operate using Interna tional Organization for

Standardization (ISO) certificati on or standards and; therefore, include internal and external

audits (ISO, 2009). ISO standards ensure desirable characteristics of seeds and services, such as
quality, safety, reliability, and efficiency. The ISO standards represent an international
consensus on good management practices with th e aim of ensuring that the organization can
consistently deliver excellent product or serv ices. The standards not only must meet the
customer’s requirements and applic able seed regulatory requirements, but must aim to enhance

customer satisfaction and achieve continual improvement of its pe rformance in pursuit of these
objectives (ISO, 2009).

Commercially certified soybean seed must meet state and federal seed standards and labeling
requirements. The Association of Official S eed Certifying Agencies (AOSCA) standards for
certified soybean seed are as follows: 98% pure seed (minimum), 2% inert matter (maximum),CBI Deleted Copy

Page 9 of 127 0.05% weed seed (maximum, not to exceed 10 per lb.), 0.60% total of other crop seeds

(maximum), 0.5% other varietie s (maximum, includes off-colored beans and off-type seeds),
0.10% other crop seeds (maximum, not to exceed three per lb.), and 80% germination and hard

seed (minimum) (AOSCA, 2009). Seed that meets or exceeds these standards are provided in
appropriately labeled seed bags to growers

Production of Soybean Grain Cont aining High Oleic Soybean Oil. Specialty soybean (e.g.

Vistive low linolenic) is currently produced under cont racts issued by processors or elevators. It
is expected that high oleic soybean will be produced using this system as well.

There are three major factors needing to be sa tisfied before a farmer will produce a specialty
soybean containing a modified soybean oil, like hi gh oleic. These factors have become evident
in Monsanto’s experience in the crop pr oduction of Vistive low linolenic soybeans.

1. Yield of the soybean must be comparable to commodity soybeans routinely planted

2. The option to market, price & deliver the grai n must be comparable to commodity grain

3. The premium paid to the farmer to offset th e cost to identity preserve the high oleic containing grain must provide incremental income opportunity above the production of commodity grain. The income opportunity cannot be realized by the fa rmer unless the grain is delivered to the processor within specifications or in its identity preserved state.
Therefore, the motivation is a financial incen tive for the farmer to avoid comingling with commodity grain, keeping all the grain identity preserved within this closed loop system of seed – farmer - processor. Many farmers willi ngly choose to plant all their acreage to this specialty soybean because it eliminates any risk of contamination from in advertent errors that may occur during the harvest. If a farmer c hooses to produce the sp ecialty grain for the processor, the farmer will arrange to store the grain on farm or at their local elevator, if the elevator is participating in the sp ecialty program with the processor.

The steps involved in secu ring grain production are:

1. Monsanto will sell and distribute high oleic soyb ean seed to the farmer after the farmer has signed and agreed to the conditions of Monsan to’s Technology and Stewardship Agreement. The agreement will mandate that the farmer sell any soybean produced into an identity preservation channe l (See Figure 2).

2. Processors will be responsible to contract w ith elevators and growers the acreage needed to fulfill the expected demand for high oleic soybean.

3. After harvest, the farmer will deliver the grai n produced to the location specified in his contract, either a participating elevator or proces sor. If to the elevator, the elevator will keep the grain segregated from commodity grain and pay the farmer his premium, provided it passes the analytical testing. Th e elevator will deliver the grain to the processor as delivery

windows and crush schedules have been established. Upon delivery of grain by the farmer, samples will be analyzed from every truc kload by Near Infrared Transmittance (NIT) developed by Monsanto. This will confirm th e grain contains high oleic soybean oil as required by the production contract . Upon confirmation of grain that meets specification of high oleic soybean grain, the processor or elev ator will approve the premium payment to the

farmer.CBI Deleted Copy

Page 10 of 127 In the event that high oleic soybean do not meet the minimum specifications established for oil quality, the soybean would be isolated from other high oleic or commodity soybean and blended with an appropriate quantity of hi gh oleic soybean to meet the specifications required for a food application. Monsanto has been conducting field trials with high oleic soybean since 2005 and has considerable expe rience with trait performance in various genetic backgrounds and under various climatic conditions. Trait performance has been consistent over several years of breeding (See Table 10). All s oybean varieties that Monsanto will introduce must meet specifications for agronomic performance and oil quality. While it is not envisioned that any of the high oleic soybean varieties will underperform, blending with other high oleic s oybean is the most likely remedy.

Grain Elevator. Grain elevators play an important role in specialty progra ms with their long
term storage of the grain. Since processing facilities crush soybean s throughout the calendar
year, soybeans used to supply these crush plants need to be stored year round. Farmers typically
prefer to empty their storage prior to planting of the new crop and prior to temperature warm up
in the spring. The warm spring and summer w eather present challenges as condensation can
build up in the bins creating moistu re related issues and grain eating insects become more active.
Farmers prefer to avoid these high management grain conditions. Commer cial elevators have
expert grain managers on staff to monitor grain quality and keep grain in condition in all weather
situations presente d throughout the year.

Commercial grain elevator s are also better equipped to ship gr ain to processors during times of
severe weather or when farmers cannot get to their bins when roads are impassable.

Processors will therefore enter into supply contracts with comme rcial grain elevators for these

reasons. Contracts will be “acre” based, establis hed to fill bin capacity that has been agreed
upon by both parties. Grain elevators will then c ontract with farmers directly, providing harvest
storage terms to the farmer with premiums to be paid that have been agreed upon with the
processor. The elevator pays the farmer the specialty grain premium upo n successful analytical
testing by NIT performed at the elevator locatio n. NIT testing equipment will be provided by
Monsanto to participating elevators identical to the equipment provided to processors.

In order for the elevator to be reimbursed fo r the premiums paid out to farmers that have
delivered to them, the elevator must in turn preser ve the identity of the grain as it is delivered to
the processor.

Every load delivered to the processor by the el evator will be analyzed using NIT technology.
Processors will approve the premium payment to the elevator after analysis of the grain confirms
high oleic soybean oil.

Processing Oil and Meal. Specialty high oleic soybean grai n will be introduced into the
processing plant as it is running commodity soyb ean grain. Upon the transition from commodity
soybeans in the plant, the processor will ensure that the equipment is lined out and operating
within normal and acceptable limits and parame ters using commodity soybean. High oleic
soybeans will then be moved through the plant c ontinuously until all specialty grain located atCBI Deleted Copy

Page 11 of 127 the plant is gone. After all of the specialty high oleic soybean s have moved through the seed
prep building, the bins and conveyors feeding the seed prep building will be filled with
commodity soybeans to keep the plant running c ontinuously. Commodity soybeans will then be
processed as normal.

Upon the transition from commodity soybeans to th e high oleic soybeans, a sample of crude oil
is required to confirm the presence of the a ppropriate fatty acid composition (FAC) unique to
high oleic oil. Until the fatty acid composition of the crude oil exiting th e extractor is of the
appropriate composition, as determined by the plant manager, off spec crude oil will be sent to a
tank that will be designated for flush oil. This will contain all oil before and after the collection
of “on-spec” high oleic oil. High oleic crude oil that is collected with the appropriate FAC will
be designated for further refining, bleaching, an d deodorization. The processor’s lab will be
used to aid in identifying the appropriate time to start collecting crude high oleic oil exiting the
extractor that will undergo further refining. Th e lab will use gas chromatography equipment to
analyze the crude oil to determine when on sp ec high oleic oil can be collected from the
extractor. A tank approved for high oleic oil will be assigned by a processor employee. After all
high oleic grain has been processed, the seed extraction area will be flushed with commodity
soybeans, and regular commodity oil will be prod uced. During the flush, crude oil exiting the
extractor will be sampled and analyzed to c onfirm the equipment has been flushed to an
acceptable level, yielding commodity soybean oil.

Processes are established by the processor to ensure the transfer of high ol eic oil to the crude oil
tank farm to minimize loss and maximize quality. The lab at the processor will utilize
appropriate analytical methods to determine when the high oleic oil is of proper quality to send
to the main crude tank(s) that will supply oil fo r further refining. Commodity soybean flush oil
prior to and after the crush of the high oleic oil will be collected in tanks designated as flush, and
will be blended with commodity soybean oil to bring the flush oil into a specification range
necessary to be sold as commodity soybean oil.

Food Company. Oil will be supplied to the food compa ny by oil processors and suppliers. The
processor or supplier will test the oil and assure that it meets specific customer requirements
including quality factors (e.g. pero xide value, oxidative stability index, color, flavor) and oil
composition (customized blends, specified fatty acid composition). Foodservice distributors
typically obtain the oil from the oil processo rs and deliver it to each foodservice outlet

(individual stores, caterers, cafeteria s, etc). From the time the oil is packaged, until it is utilized
at the specific customer’s facility, there is pr oper identification of th e oil through labeling and
manufacturing codes allowing for sufficient produc t traceability if needed. Individual facilities
will utilize proprietary inventory and ordering sy stems that are in place to insure that the
appropriate oil is ordered, delivered and utilized.

High oleic soybean oil will be used by the food i ndustry in several ways. One specific type of
food company customer will be foodservice operato rs including quick service restaurants, casual
dining and full service restaurants who will use hi gh oleic soybean oil as a fry medium and inCBI Deleted Copy

Page 12 of 127 meal preparation. Food companies will also use hi gh oleic soybean oil as a food ingredient in a
range of food products including as spray oil on crackers and snacks, or as a component of oil
blends used in shortenings and other foods. Each individual f ood company has in place systems
for ingredient (oil) ordering, re ceipt, storage, access and lo t identification at specific
manufacturing locations, as well as finished pr oduct (the food which incorporates high oleic
soybean oil) batch identification, manufacturing facility, storage, shipment to distribution
centers, customer order picking, cu stomer order shipment and rece ipt. Appropriate procedures
are currently in place to insure traceability from receipt of the ingredient through distribution to a
specific retailer’s facilit y. Food companies routinely conduct mock recalls including use of the
media should the need arise. Food manufacturing facilities also comply with federal and state
requirements for good manufacturing practices and product traceability. Supply chain
consultants can be employed to confirm appropria te systems have been established that meet
ingredient and product traceability requirements.



### Stewardship of High Oleic Soybean

Monsanto is committed to product stewardship and to implementing BIO’s "Excellence through
Stewardship" program and Pr oduct Launch Stewardship Policy 5 . Monsanto considered Annex 2
“Special Use traits in Commodity Crops” 6 to develop launch plans for high oleic soybean oil
including: (1) identifying relevant stakeholde rs for the trait and cr op and engaging them in
dialogue regarding use of high olei c soybean oil and potential imp acts to vegetable oil markets,
(2) conducting a market and trade assessment, in cluding securing regulato ry approvals in key
export countries prior to full co mmercial launch, (3) developing a risk mitigation plan, and (4)
undertaking appropriate outreac h, necessary to educate stak eholders and implement the
management plan for high oleic soybean oil. These actions protect agai nst adverse impacts to
trade of soybean due to the introduction of a new biotechnology improved soybean.

Stakeholder Dialogue
Monsanto is committed to dialogue with key industry stakeholder groups and has held several
meetings with the National Oil Processors A ssociation (NOPA) as we ll as other key industry

associations such as the North American E xport Grain Association (NAEGA), National Grain

and Feed Association (NGFA), North American Milling Association (NAMA), American Bakers

Association (ABA), and Gro cery Manufacturers Association (GMA). Soybean grower
organizations: American Soybean Association (A SA), United States Soybean Board (USB), and
many state soybean associations. In addition QUALISOY, a collaborative program sponsored by
the USB that serves as an independent, third pa rt resource for information on trait-enhanced
soybean oils, has been kept informed on the plans for this product, along with leaders in dietary

and nutrition fields.

5 http://www.monsanto.com/ourcommitmen ts/Pages/product-stewardship.aspx

6 http://www.bio.org/letters/Product _Launch_Stewarship_12_10_09.pdf ;CBI Deleted Copy

Page 13 of 127 Market and Trade Assessment
Monsanto has conducted a market and trade assessment to determine the impact of the
introduction of high oleic soybean oil. Soybean is a globally traded commodity with the U.S.
being the top global producer (Soyatech, 2010). Biotechnology-de rived crops and their use as
food and feed are subject to regulation in many c ountries. In order to su pport continued trade in
soybean, Monsanto is pursuing regulatory a pproval for MON 87705 in all key soybean import
countries with a functioning regulatory system to support the flow of international trade.
International regulatory authoritie s are evaluating the biotech com ponent as well as the modified
oil fraction. It is expected that uses of high oleic oil soybean will be similar on a global basis.

There would be no impact to human heath or to the use of any of the other processed fractions
produced from soybean due to comingling of hi gh oleic soybean with commodity soybean.
Monsanto has completed a GRAS assessment of the high oleic soybean oil (MON 87705 in a
Vistive background) 7 and has completed the biotechnology consultation on MON 87705 with the

U.S. FDA 8 . The GRAS assessment has been evaluated by FDA and the agency had no further
questions. The biotechnology consultation consider ed the food and feed safety impacts due to
the genetic modification process, RNAi suppr ession of two endogenous enzymes resulting in
modification to fatty acid metabolism, as we ll as exposure to the CP 4 EPSPS protein. The
GRAS assessment considered the nutritional imp act of total replacement of commodity soybean
oil with high oleic soybean oil. High oleic soybean oil is sim ilar to other high oleic vegetable
oils that are commonly consumed producing no harm ful effects to humans. Hence, Monsanto as
well as a panel of qualified scientific experts have concluded that high oleic soybean oil is
generally recognized as safe.

Information provided to USDA showed that the impact due to the suppression of the two
endogenous enzymes is restricted to seed. With the exception of the intended changes in fatty
acid composition and presence of the CP4 EPSPS pr otein, the soybean meal and other processed

fractions used for animal feed and human food applications are compositionally equivalent to
commodity processed soybean fractions 9 . Monsanto provided information to USDA in the
petition demonstrating that the composition of the meal was compositionally equivalent to meal
derived from conventional soybean and safe an d wholesome for food or feed applications.

Monsanto has assessed the impact of the introduction high oleic soybean oil on commodity and
other vegetable oils. In response to stakeholde r dialogue, Monsanto was specifically requested
by NOPA to address the impact of comingling of commodity soybean oil with high oleic
soybean oil to bottled oils (100% commodity s oybean oil) and severe heat processing (e.g.
impact to frying applications and sensory proper ties of prepared foods). This assessment has
been shared with key stakeholders such as NOPA, NAEGA, ASA, Qualisoy, USB and GMA.

7 http://www.fda.gov/Food/GuidanceComplianceRegulatoryI nformation/GuidanceDocuments/FoodIngredientsandPa ckaging/ucm061846.htm#Q1

8 Biotechnology notice: BNF 121: http://www.accessdata.fda.gov/scripts/fc n/fcnNavigation.cfm?rpt=bioListing ; GRAS notice: GRN 306 - http://www.acces sdata.fda.gov/scripts/fc n/fcnNavigation.cfm?rp t=grasListing&page=2

9 USDA Petition Number 09-201-01p, Section VII.C.CBI Deleted Copy

Page 14 of 127 Hypothetical impacts were assessed using the foll owing scenario: (1) hi gh oleic soybean grain
or oil went unnoticed and was comingled ( e.g. harvest mistake, inadvertent mixing of grain
during transport, mistaken deliver y of grain, etc...), (2) the comingl ed soybean were delivered to
an oil processor and mixed with commodity grain or soybean oil, (3) th e oil or grain was not
analyzed for oil composition by anyone in the food supply chain, and (4) the comingled oil was
used in the food or feed supply chain. This s cenario is highly unlikely given existing critical
control points embedded in the production handling and processing system and because high
oleic soybean oil is expected to be produced in a contractual identity preserved system in order to
preserve its premium value. Processors will or iginate the supply of high oleic soybeans and as
they have extensive experience managing, segr egating, testing, formulat ing and blending other
sources of high oleic vegetable oils such as high oleic canola and high oleic sunflower oil.

Bottled vegetable oil is not an intended use for high oleic soybean oil. However, bottle oil was
used to assess potential impacts at the request of NOPA. It re presents a readily measurable
impact that could occur if hi gh oleic soybean were comingled with 100% commodity soybean.
Mixing high oleic soybean with commodity soybean will impact to the leve ls of fatty acids in
commodity soybean. This effect is measurable and can potentially translate into downstream
effects to food labeling, oil quality, and functionality. Even though such comingling is
improbable, potential impacts were assessed at several different comingling levels ranging from
4% and up to 30% using four different paramete rs: (1) food ingredient labeling, (2) nutritional
facts panel labeling (3) func tionality of the oil, and (4) sensory evaluation.

Food Ingredient Labeling Codex Specification. Effects were assessed on the ability to meet
USP Food Chemical Codex (USP FCC) soybean o il labeling specification. In Table 3 the
impact of varying amounts of high oleic soybean oil comingled with commodity soybean oil
on the ability to meet specifications for major fatty acids is evaluated. The USP FCC is a
compendium of internationally recognized standards for the purity and identity of food
ingredients and is used as a set of agreed standards between buyers and manufacturers of
food ingredients 10 . Based on this analysis the most se nsitive fatty acid is 18:2 linoleic acid.
If high oleic soybean were co-mingled with co mmodity soybean oil at levels above 14%, the

linoleic acid (C 18:2) content of the resulting oil following processing would fall below the
USP FCC specification for commodity soybean o il. At levels above 15% oleic (C 18:1),
linoleic acid would be out of the sp ecifications for soybean oil.

To put this into perspective, 15% comingli ng could occur due to a farmer mistakenly
harvesting approximately three acres of high oleic soybean (assuming yield = 43
bushels/acre) and comingling it with commod ity soybean in a grain truck hauling the
standard load limit with a capac ity of 900 bushels. If the grai n were delivered to a grain
elevator or directly to a pr ocessor, it would be mixed with other commodity soybean grain
thereby diluting the high oleic soybean consider ably with commodity soybean. The capacity
of grain elevators or bin storag e tanks is highly va riable; however, bin capacities up to and

10 http://www.usp.org/fcc/CBI Deleted Copy

Page 15 of 127 exceeding 1⁄2 million bushels are quite common. Hence, it would take roughly 83 fully
loaded trucks containing 100% high oleic soyb ean or multiple mistakes during harvest of
high oleic soybean to achieve a 15% level of comingling.

Nutrition Facts Panel Labeling. Figure 3 and 4 demonstrate the amount of high oleic
soybean oil that would be required to be comi ngled with commodity s oybean oil in order to
impact the Nutrition Facts Panel for major fatty acid categories. Based on this analysis,
monounsaturated fat is the most sensitive fatty acid category and comingling up to 5% high
oleic soybean oil could be tolerated befo re the Nutrition Facts Panel label for
monounsaturated fatty acids were impact ed on retail bottled vegetable oil. Up to 15% high
oleic soybean could be comingled with co mmodity soybean oil before effects to
polyunsaturated fatty acid labe ling would be impacted.

The threshold for effects is lower than USP FC C labeling situation discussed above. In this
case it would take a mistake during harvest of one acre of high oleic soybean comingled with
900 bushels of commodity soybean in a 900 bushe l grain truck or 28 fully loaded trucks
containing 100% high oleic soybean would need to be delivered to a 1⁄2 million bushel grain
elevator containing commodity soybean to trigge r an effect to the nutritional facts panel
labeling.

The impact due to both of the comingling scenar io discussed above would be minimal. As
discussed previously, there would be no impact s to human health and the changes in fatty

acids would benefit consumers from a nutritio nal perspective. Mandatory labeling is
required for saturated fats, in this case coming ling levels of up to 28% could be tolerated
without impacting the level of saturated fats re ported on the label. At 28%, the level of
saturated fat would be lower, enhancing the nutritional profile of th e product (Figure 4).

Oil Functionality; Severe Heat Processing. High oleic soybean oil has an improved stability
profile compared to commodity soybean oil. An assessment of the impact of comingling of
high oleic soybean oil with commodity soybean grain and oil was conducted. Information in
Figure 5 demonstrates that comingling of high oleic soybean oil with commodity soybean oil
increases the OSI stability index for commodity s oybean oil. This outcome is expected since
soybean oil oxidative stability is drastically influenced by the proportion of monounsaturates
to polyunsaturates, and high olei c soybean oils are estimated to have improved oxidative
stability compared to conventional soybean oil (Frankel, 2005).

Food Sensory Assessment . A sensory assessment of high ol eic soybean oil was performed to
evaluate consumer acceptability of high oleic soyb ean oil. Figure 6 demonstrates the sensory
results for high oleic soybean oil in the most challenging environment high temperature food
frying. In this experiment, commodity soybean oil and blends (5% to 15%) of commodity
and high oleic soybean oil were used to prepar e French fries. Food testers were asked to
evaluate the quality of the fries using the Sensory Quality System described by King et al
(2003). The outcome from the sensory eval uation showed that high oleic soybean oil
comingled with commodity soybean oil blends were essentially the same as commodityCBI Deleted Copy

Page 16 of 127 soybean oil over the six-day period during wh ich the experiment was conducted. Thus,
comingling of high oleic soybean oil with comm odity soybean oil would have no impact to
the sensory profile of commodity soybean o il should inadvertent co mingling occur (Figure

6).

The comingling scenarios and impacts above are pres ented as the most conservative scenarios.
Oilseed processors and users of vegetable oils are accustomed to the presence of numerous
vegetable oils of differing fatty acid makeup that are available concurrently for blending and use
in various food applications. The comingling leve ls described here where an impact could occur
in functionality or la beling would happen only in instances where common control points were
ignored. Such levels of comingling are highly unlikely to occur due to economic incentives to
growers, legal contracts, stew ardship SOP’s (seed quality to end user), and demonstrated
competency in managing inadvertent comingling with other vegetable oils that have a fatty acid
profile similar to high oleic soybean oil. In a ddition, fatty acid analytic al methods are widely
available and used currently by oil pr ocessors and food manufacturers.

Industry Outreach

Monsanto has held conversations with the soybean and food industry key stakeholders
mentioned above regarding the oil composition, stewardship plan and performance of MON
87705. Additionally, Monsanto has consulted wi th NOPA and industry members of NOPA’s
biotechnology committee as well as the ASA and USB regarding the fatty acid composition of
high oleic soybean oil and potential changes to commodity soybean oil due to comingling.
NOPA and members of their biotechnology committee were provided information related to the
fatty acid composition of high ol eic soybean oil derived from MON 87705 in the Vistive lowlinolenic genetic background as well as oil prope rties and mixing effects information described
previously. NOPA and biotechnology committee members agree that potential impacts related
to the unintended mixing of commodity soybean oil and high oleic soybean oil could be
remedied through blending, a common industry practi ce. Given the control points in the system
and potential market impact of high oleic soybean oil, a risk mitigation plan (as described in
Annex 2 of BIO’s Product Launch Stewardship Polic y) for high oleic soybean is not warranted.
NOPA and the committee members continue to provi de input on various aspects of the intended

commercialization.



### Summary

High oleic soybean offers an opportunity for soyb ean growers to recapture markets for vegetable
oil previously occupied by soybean oil and cr eate added value for U.S. soybean producers.
Given the abundance of vegetable oils on the market and demonstrated ability of the system to
adapt to consumer preferences incorporating new oils in to existing food manufacturing
processes, the market impact of high oleic soybean is expect ed to be minimal and easily
managed. Monsanto has conducted and is implemen ting a product stewardship plan that is basedCBI Deleted Copy

Page 17 of 127 upon consideration of BIO’s Launch Stewardship guidance and upon experience with previous
successful product introductions completed by Monsanto. Mons anto continues to engage

stakeholders and educate them on the benefits of high oleic soybean and proper stewardship
practices.CBI Deleted CopyPage18of127Table 4. Impact of comingling of higholeic soybean oil on Food Codex specifications of commodity soybean oil.1FCC – Food Chemical Codex specification for named vegetable oils2HO – Oil profile represents high oleic MON 87705 soybeanbred into a low linolenic soybean background.Legend. The impact of mixing high oleic soybean oil on the fatty acid profile of commodity soybeanoil is depicted in the table above.The impact to the fatty acid profile was assessed at variouspercentages of high oleic soybeanoil mixed with commodity soybeanoil.The “break point” reflects the percentageof high oleic soybean oil at which the fattyacid levels in soybean oil would no longer be inthe USP FCC specification range for soybean oil.CBI Deleted CopyPage19of127Figure 3. Impact of comingling high oleic soybean oil with bottled commodity soybeal oil on nutritional facts labeling to monoand polu unsaturated fatty acids.Legend. The impact of mixing high oleic soybeanoil on nutritional facts labeling for bottled soybean oilis depicted in the figureabove. The impact to nutritional facts panel labeling was assessed at various percentages of high oleicsoybean oil mixed withcommodity soybean oil. The blue arrows indicate the levelsof high oleic soybean oil that would impact the nutritional factslabeling.CBI Deleted Copy

Page 20 of 127 Figure 4. Impact of comingling high oleic soybea n oil with bottled commodity soybeal
oil on nutritional facts labeling to saturated fatty acids.

Legend. The impact of mixing high oleic soybean oil on nutritional facts labeling for bottled
soybean oil is depicted in the figure above. The impact to nutritional facts panel labeling was
assessed at various percentages of high oleic soybean oil mixed with commodity soybean oil.
The blue arrows indicate the levels of high ol eic soybean oil that would impact the nutritional
facts labeling for mandatory fatty acid reporting (saturated fats).CBI Deleted Copy

Page 21 of 127 Figure 5. Oxidative stability index of high ol eic soybean blended with commodity soybean

oil.

Legend. The impact of comingling of high olei c soybean oil was assessed on the oxidative
stability index of the blended oil. The OSI stabilit y index is an indicator of soybean oil stability.

All oils and fats have a resistance to oxida tion, which depends on the degree of saturation,
antioxidant and prooxidant concentration, and prior a buse. Oxidation is slow until this resistance
is overcome, at which point oxidation accelerates and becomes more rapid. The length of time
prior to the acceleration of oxidati on is referred to as the ‘induction period,’ and the point of
maximum rate change is referred to as the Oxidat ive Stability Index or Oi l Stability Index (OSI),
and is reported in hours (Frankel, 2005). 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

0 10 20 30 40 50 60 70 80 90 100OSI@110oC

% High Oleic in Commercial Soy OilCBI Deleted CopyPage22of127Figure 6. Sensory assessment ofhigh oleic soybean oil compared to other vegetable oils.Legend. A sensory assessment of the quality ofFrench fries prepared in blends ofhigh oil/commodity soybean oil was conductedover a period of six days. A modificationof the procedure describedby King et al (2002) was usedfor the evaluation. The sensoryqualities of the oil blends tested were essentially the same over the six-day period.Scale: 5 = no difference, 4 = very slight difference,3 = slight difference, 2 = different, 1 = very different.CBI Deleted Copy

Page 23 of 127 References

AOSCA 2009. Seed Certification Handbook. Associa tion of Official Seed Certifying Agencies.

Moline, Illinois.

Delaney, B., Appenzeller, L. M., Munley, S. M., Hoban, D., Sykes, G. P., Malley, L. and
Sanders, C. (2008) Subchronic feeding study of high oleic acid soybeans (Event DP-3Ø5423-1)

in Sprague-Dawley rats, Food and Chemical Toxicology

, 46 , 3808-3817.

Frankel, E.N., 2005. Pages 21, 201-205 in Lipid Ox idation. The Oily Press, Bridgewater,
England.

Graef, G., LaVallee, B.J., Tenopir, P., Tat, M., Schweiger, B., Kinney, A.J., Van Gerpen, J.H.
and T.E. Clemente. 2009. A high-oleic-aci d and low-palmitic-acid soybean: agronomic
performance and evaluation as a feedstock for biodiesel. Plant Biot echnology Journal, 7:411-

421.

ISO. 2009. Selection and Use of the ISO 9000 Family of Standards. Inte rnational Organization
for Standardization. http://www.is o.org [Accessed March, 2010].

King. S., Gillette, M., Titman, D., Adams, J ., and M. Ridgely. 2002. The sensory quality
system: a global quality cont rol system. Food Quality and Preferences, 13: 385-395.

Möllers, C. (2004) Potential and fu ture prospects for rapeseed o il. In: Gunstone, F. D. (ed.)
Rapeseed and Canola Oil. Producti on, Processing, Properties and Uses . CRC Press LLC, Boca
Raton, FL, pp. 186-217.

Soyatech. 2010. Statistics. Soyatech, Manitoba, Canada.

Wilson, 2004. Seed composition. P. 621-629. In Boerma, H.R. and Specht, J.E. (eds.) Sopybeans: Improvement, production and uses . ASA-CSSA-SSSA, Ma dison, Wisconsin. 1144

pp.CBI Deleted Copy

Page 24 of 127 

# ATTACHMENT I

THE MARKET POTENTIAL FOR A HIGH OLEIC SOYBEAN OIL

CONFIDENTIAL BUSI NESS INFORMATIONCBI Deleted Copy

Page 25 of 127 [

CBI Deleted

Pages 25 through 127 have been deleted

]