1 

## PLANT PEST RISK ASSESSMEN T FOR MON 87769 SOYBEAN

Monsanto Company (Monsanto) has petitioned APHI S for a determination that the genetically engineered (GE) stearidonic acid soybean ( Glycine max ) event MON 87769 ( hereafter referred

to as MON 87769 ) is unlikely to pose a plant pest risk (Monsanto 2010) and, therefore, is no longer a regulated article under the APHIS’ 7 Code of Federa l Regulations (CFR) part 340. APHIS administers 7 CFR part 340 under the authority of the plan t pest provisions of the Plant

Protection Act of 2000 1 . This plant pest risk assessment was conducted to determine if MON 87769 is unlikely to pose a plant pest risk.

MON 87769 was produced by transforma tion of soybean tissue using Agrobacterium tumefaciens

, a plant pest, and some of the sequences used in the transformation process are also from plant pests, including some either retained in MON 87769 (i.e., tml terminator sequence and left and right T- DNA border sequences from A. tumefaciens ) or eliminated (35S RNA from figwort mosaic virus); therefore, the engineered soybean has been considered a regulated article under APHIS regulations at 7 CFR part 340.

Potential impacts to be addressed in this risk a ssessment are those that pertain to the use of MON 87769 and its progeny in the absence of confin ement. APHIS uses data and information submitted by the applicant, in addition to current literature, to assess if MON 87769 is unlikely to pose a plant pest risk. If APHIS determines that a GE organism is unlikely to pose a plant pest risk, then APHIS has no regulatory authority over that organism.

Potential impacts in this Plant Pest Risk Assessm ent are those that pertai n to plant pest risk characteristics. APHIS regul ation 7 CFR 340.6(c) specifies the information needed for consideration in a petition for nonregulated st atus. APHIS will evaluate information submitted by the applicant related to plant pest risk characteristics, dise ase and pest susceptibilities, expression of the gene product, new enzymes, or changes to plant metabolism, weediness of the regulated article, any impacts on the weediness of any other plant with which it can interbreed, and transfer of genetic info rmation to organisms with wh ich it cannot interbreed.

An analysis of agricultural or cultivation practices and effect s on other agricultural products associated with MON 87769, and impacts of MON 87769 on the environment will be considered in the Environmental Assessment (EA) for MO N 87769. Likewise, a thorough assessment of the effects of the determination on nontarget organism s, beneficial organisms, and threatened and endangered species will be considered in the EA.

1 Section 403 (14) of the Plant Protection Ac t (7USC Sec 7702(14)) defines plant pest as: “Plant Pest - The term “plant pest” means any living stage of any of the following that can directly or indirectly injure, cau se damage to, or cause disease in any plant or plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A f ungus. (F) A virus or viroid. (G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the articles speci fied in the preceding subparagraphs.”2 

## Introduction

a. History of Development of MON 87769 Stearidonic Acid (SDA) Soybean
Soybean has been cultivated extensively and improved through conventional breeding programs following its introduction in the U. S. and subsequently has become a key source of nutrients for

food and feed use in the U.S. (Hymowitz and Singh 1992). Monsanto developed SDA MON 87769 by using recombinant DNA technology to geneti cally engineer (GE) into a conventional soybean variety two foreign desaturase genes, Primula juliae Δ 6 desaturase ( Pj.D6D) and Neurospora crassa Δ 15 desaturase ( Nc.Fad3), whose combined action results in the production of two fatty acids, stearidonic acid (SDA) and γ - linolenic acid (GLA), that do not occur in conventional soybeans (Figure 1 below, adapted from Figure VII-1 of Monsanto 2010, p. 103).

Δ 9 Desaturase Δ 12 Desaturase Δ 15 Desaturase

18:0 18:1 18:2 18:3

stearic acid ol eic acid linoleic acid α -linolenic acid (LA) (ALA)

Δ 6 Δ 6

Desaturase Desaturase

Δ 15 Desaturase

18:3 18:4

γ -linolenic stearidonic

acid acid (GLA) (SDA)

Pathways where soybean endogenous desaturases work Pathways where exogenous (engineered) desaturases work

Figure 1. Fatty acid biosynthesis in plants and the introduced changes to changes to produce MON 87769 Soybean (adapted from Monsanto 2010, Figure VII-1)

MON 87769 seed oil consists of approxima tely 20-30 percent stearidonic acid (SDA), a

sustainable alternate source of an omega-3 fatty acid to help meet the needed dietary intake of long chain omega-3 fatty acids (Harris et al. 2008 ). SDA is an omega-3 polyunsaturated fatty acid (PUFA) composed of an 18-carbon chain w ith four double bonds (1 8:4). Although SDA is found in fish, fish oils, green leafy vegetables , and some nuts and vegetable oils, it occurs relatively high levels in only a limited number of fish, and seaweed, and rarely in other plant species used as food sources (Figure 2 in Gu il-Guerrero 2007; Whelan 2009). In mammals, SDA is a metabolic intermediate in the producti on of the longer car bon chain omega-3 PUFAs eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) from alpha linolenic3 acid (ALA; 18:3), a common dietary constituent of vegetable oils . In addition to SDA, MON 87769 seed oil contains approximately 7% γ - linolenic acid (GLA; 18:3), an omega-6 PUFA

which is an in vivo metabolite in the conversion of linoleic acid (LA: 18:2) to arachidonic acid (20:4) in mammals (Monsanto 2010, p. 4). A lthough the therapeutic and health-promoting effects of consuming omega-3 long chain PUFAs from fish (SA, EPA, and DHA) are widely recognized, typical Western diets contain very little fish, and the same benefits may not be shared by their more commonly consumed precursor alpha linolenic acid (ALA). In contrast, comparative results suggest that SDA shares many of the biological effects of the omega 3 longer chain PUFAs and functions most similarly to dietary EPA, compared with ALA, when consumed in a typical Western diet (Whelan 2009). However, the lo wer number of double bonds in SDA compared to EPA and DHA makes it more stable to oxidation (less prone to fishy or rancid orders and taste) which is expected to expand its use as an alternative to fish oil in food products. While 6% of the world’s fish oil is used for human consumption and an even amount for animal feed, 87 % is used in fish feed la rgely to support aquacultu re (Whelan 2009). Oils enriched in SDA (such as those from Echium ) or SDA itself have been shown to provide similar results as fish oil with respect to growth and f eed efficiency in some fi sh species (e.g. Arctic char, cod, Atlantic salmon, and hybrid striped bass) (Bharadwaj et al 2010). According to Monsanto, MON 87769 can serve as an alternate source to help meet the needed dietary intake of long chain omega-3 fatty acids in food and animal feed (poultry and aquaculture).



### b. Description of the In serted Genetic Elements
MON 87769 was developed through A. tumefaciens mediated transformation of soybean line A3525, a nontransgenic, mid-maturity group III, conventional variety with very high yield potential (Steffen 2004). The disarmed A. tumefaciens used for the transformation harbors a plasmid vector PV-ZMPQ1972 that contains two separate T-DNAs (Monsanto 2010, Figure IV1, p. 45). The first T-DNA, designated as T-DNA I, contains two expression cassettes: Primula juliae Δ 6 desaturase ( Pj.D6D ) gene expression cassette and Neurospora crassa Δ 15 desaturase 3 ( Nc.Fad3) gene expression cassette. The sec ond T-DNA region (T-DNA II) contains the cp4

epsps gene expression cassette that encodes the CP4 EPSPS protein (5 -enolpyruvylshikimate-3- phosphate synthase protein from Agrobacterium sp. strain CP4) that provides tolerance to the action of glyphosate, which is the active ingred ient in Roundup® agricu ltural herbicides.

T-DNA I region incorporated into MON 87769 c ontained the following genetic elements (Monsanto 2010, Table V-1, p. 50):

 Border Sequence: Left and ri ght T-DNA border sequences from A. tumefaciens to facilitate T-DNA transfer (Depicke r et al. 1982; Barker et al. 1983).

 7S α Promoter: Promoter and leader from the Sphas1 gene of soybean encoding betaconglycinin storage protein (D oyle et al. 1986) that directs mRNA transcription in seed.

 Δ 6D gene: Δ 6d gene coding sequence from Primula juliae (Ursin et al. 2008) that codes

for Δ 6 desaturase, which creates a double bond at the 6 th position from the carboxyl end of a fatty acid.

 tml terminator: 3 ′ untranslated region of the tml gene from A. tumefaciens octopine-type Ti plasmid (Kemp et al. 2000).

 7S α promoter: Promoter and leader from the Sphas2 gene from soybean encoding the alpha subunit of beta-conglyc inin storage protein (Wang and Dubois 2004) that directs mRNA transcription in seed.4  Fad (fatty acid desaturase) 3 gene: Fad3 gene coding sequence from Neurospora crassa (Ursin et al. 2006) that codes for Δ 15 desaturase, which crea tes a double bond at the 15 th
position from the carboxyl end of a fatty acid.

 E-9 terminator: 3 ′ untranslated region of the p ea ribulose 1,5-bisphosphate carboxylase small subunit 2 ( rbcS2) gene which functions to dire ct polyadenylation of the mRNA (Coruzzi et al. 1984). In addition to the above-mentioned genetic el ements, the insert also contained noncoding intervening sequences of 8-101 base pair length that were us ed for DNA cloning.

T-DNA II region, which was bred out of the final MON 87669 event , contained the following genetic sequences (Monsant o 2010, Figure IV-1, pp. 45-47):

 Border Sequence: Left and ri ght T-DNA border sequences from A. tumefaciens to facilitate T-DNA transfer (Depicke r et al. 1982; Barker et al. 1983).

 FMV promoter: Promoter for the 35S RNA fr om figwort mosaic virus (FMV) (Rogers, 2000) that directs transc ription in plant cells.

 ShkG leader sequence: 5' untransla ted leader sequence from the Arabidopsis ShkG gene encoding EPSPS (Klee et al. 1987) that helps regulate gene expression.

 CTP2 coding sequence: Transit peptide region of Arabidopsis thaliana EPSPS (Klee et al. 1987) that directs trans port of the CP4 EPSPS protei n to the chloroplast.

 cp4 epsps coding sequence: Codon modified coding sequence of the aroA gene from the Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein (Barry et al. 1997; Padgette et al. 1996).

 E9 terminator: 3' untranslated region of the pea rbcS2 gene which functions to direct polyadenylation of the mRNA (Coruzzi et al. 1984).

Monsanto provided evidence demonstrating that,

 the A. tumefaciens strain ABI that was used to transform MON 87769 was made nonpathogenic by removing the pathogenic se quences encoding phytohormones that cause crown gall tumors from the Ti (t umor inducing) plasmid present in the Agrobacterium (Koncz and Schell 1986);

 the final product does not contain any of th e backbone sequences outside of the T-DNA I

borders from the transformation vector , PV-GMPQ1972 (Monsanto 2010, Figures V-3, p. 53; V-5, p. 59);

 the final product does not contain T-DNA II elements that contain the cp4 epsps gene (Monsanto 2010, Figure V-6, p. 62);

 the DNA inserted into the soybean genome is pr esent at a single locus and contains one functional copy of D6D (Monsanto 2010, Figure V-8, p. 69) and fad3 (Monsanto 2010, Figure V-11, p. 72) genes;

 the sequence and organization of inserted genes in MON 87769 are identical to their original sequences and arrangements in the donor plasmid PV-GMPQ1972 (based on the DNA sequence alignment between the MON 87769 insert sequence to the PVGMPQ1972 transformation vector sequen ce) (Monsanto 2010, Figure V-3, p.53 and p. 81);

 no novel open reading frames were created that spanned either the 5 ′ or 3 ′ junctions between the T-DNA and soybean genomic sequences;5  the stability of the introduced genes was demonstrated by the presence of introduced genes of T-DNA I via Southern blot fingerprint of MON 87769 (Monsanto 2010, Figures V-14 and V-15, pp. 78-79) from five segregating gene rations tested (Monsanto 2010, Figure V-13, p. 77) in the breeding history. The stability was further confirmed by

the Mendelian inheritance of the T-DNA I in MON 87769 (Monsanto 2010, Table V-3, p. 84) in three segregating generatio ns (Monsanto 2010, Figure V-17, p. 83).

Because the T-DNA II region, containing the herbicid e resistant gene cp4 epsps and associated regulatory elements, has been removed from the final MON 877769 event seeking deregulation, those genetic sequences are excluded from the plan t pest risk assessment discussion presented in the following paragraphs.



## PLANT PEST RISK ASSESSMENT

Based on information on the biology of soybean (OECD 2000), data presented by Monsanto Company (Monsanto 2010), and scient ific data relevant to the di scussion of plant pest risk, APHIS makes the following conclusions for MON 87769:



### Potential Impacts of Genetic Modificati ons on Disease and Pest Susceptibilities
USDA-APHIS assessed whether MON 87769 soybean is likely to have significantly increased disease and pest susceptibility. This assessm ent encompasses a thorough consideration of introduced traits, their impact on agronomic traits and plant composition, a nd interactions with pests and diseases.

Monsanto used well-established qualitative or quantitative techniques (Monsanto 2010, Appendix I, pp. 344-346) to measure field trials for damage due to diseases (Alternaria leaf spot, Anthracnose, Asian rust, Bacterial blight, B acterial pustule, Brown spot, Brown stem rot, Cercospora leaf blight, Charcoal rot, Downy mildew, Frogeye leaf spot, Fusarium

, Phytophthora , Powdery mildew, Pythium

, Rhizoctonia

, Sclerotinia

, Septoria , Soybean mosaic virus, Soybean rust, Stem canker, Sudden death, White mold) and in sect pests (Aphid, Bean leaf beetle, Blister beetle, Corn rootworm, Flea be etle, Grasshopper, Green cloverworm, Japanese beetle, Leafhopper, Leafroller, Mexican bean be etle, Spidermite, Seed corn maggot, Soybean stem borer, Stink bug, Tarnished plant bug, Thistle ca terpillar, Thrips, Velvetbean caterpillar, White fly, Wireworm, and Woolybear caterpilla r) for two years conducted across different agroecological conditions. Data were collected on pest and di sease damage across 17 sites in 2006 and 4 sites in 2007, and 4 sites in 2006 and 2007 were quantitatively tested for abundance of arthropods. There was a slight difference in sus ceptibility to frog eye leaf spot at one site in 2006. There was a significant difference (increase) in the abundance of tarnished plant bug and two beneficial arthropods at one site (IL1) during one collec tion point and for leafhopper and stinkbug at one collection point at one location (MI). However, these differences were either within the range of the referen ce varieties and /or were not found to be significantly different in other locations. The data submitted by Monsanto indicated no meaningful differences between MON 87769 soybean and the non-transgenic counterparts for diseases (Monsanto 2010, Appendix Tables I-3 and I-4, pp. 349-350), or insect pests (Mons anto 2010, Appendix Tables I-5 to I-7, pp. 351-355; Table I-9, pp. 358-360). Thus MON 87769 is expected to be susceptible to the same plant pathogens and inse ct pests as conventional soybean.6 Monsanto’s aforementioned data (expression of the gene products, nutri tional and compositional analyses) indicate that MON 87769 is not biologically different from conventional soybean (with the exception of the Δ 6D and Δ 15D proteins) and modified fatty acid phenotypes did not alter the pest and disease incidences on Mon 87769; th erefore, MON 87769 is no more susceptible to pests and diseases compared to conventional soybean cultivars.



### Expression of the Gene Product, New Enzy mes, or Changes to Plant Metabolism

The Δ 6D protein expressed in MON 87769 is nearly id entical to the native protein produced by Primula juliae containing 446 amino acids and three histid ine motifs that are characteristic of integral membrane desaturases (Monsanto 2010, Figure VI-1, pp. 87-89; Ursin et al. 2008). Furthermore, the petition er’s analysis of the Δ 6D deduced amino acid sequence has shown that,

the MON 87769 Δ 6D is similar to other Δ 6 desaturases in ami no acid composition and enzymatic structure (Nakamura and Nara 2004). Likewise, the Δ 15D protein produced in MON 87769 is identical to the native protein produced by Neurospora crassa , with the exception of a single amino acid change from threonine to alan ine at the first amino acid after the start codon. The petitioner deliberately introduced this change to facilitate the insertion of the gene into the plant transformation vector. The MON 87769-expressed Δ 15D protein consists of 429 amino

acids and three histidine motifs that are char acteristic of integral membrane desaturases (Monsanto 2010, Figure VI-3. p. 89).

The functional activities of the Δ 6D and Δ 15D proteins were confirmed in vivo after expression from the yeast expression vector in Saccharomyces cerevisiae (see Monsanto 2010, pp. 87-90 for details). Monsanto collected ti ssue samples from leaf, forage, root, mature seed and immature seed to quantify Δ 6dD and Δ 15D protein expression in MON 87769 (see Monsanto 2010, Appendix D for the protein expression analysis , pp. 237-239). Because the expression of the Pj Δ 6D and Nc Δ 15D proteins is driven by a 7S α ' and 7S α seed specific promoter, respectively, those two proteins were not dete cted in leaf and root tissues of MON 87769, while both proteins

were detected in immature seed and at much lower levels in mature seed and forage, which

contains a small of amount of residual imma ture seeds (Monsanto 2010, Tables VI-1, p. 95 and VI-2, p. 96). The highest levels of Pj Δ 6D and Nc Δ 15D proteins measured in immature seeds of

MON87769 were 210 and 330 μ g/g of tissue on a dry weight (DW) basis, respectively, and in the mature seed they represent no more than 0.00043% and 0.00239% of the total protein. As expected, the Δ 6D and Δ 15D proteins were not detected in the conventional soybean control, A3525 (Monsanto 2010, p. 94).

USDA-APHIS assessed whether changes in pl ant metabolism or composition in MON 87769 is likely to alter plant pest risk. The assessmen t encompasses a consider ation of the expressed protein or enzymes and their effect on plan t metabolism and an evaluation of whether the

nutrients and antinutrient levels in harveste d seed and forage deri ved from MON 87769 are comparable to those in the conventional soybean control, A3525.

Detailed compositional and nut ritional comparisons of MON 87769, the conventional soybean control A3525, and ten commercially available soybean varieties were conducted on samples

collected from fives sites across the U.S. in 2006. The analysis included protein, fat, carbohydrates, fiber, ash, moisture, amino acids, fa tty acids, vitamin E, and antinutrients and key7 secondary metabolites, consistent with OECD guidelines (OECD 2001). Appendix E (Monsanto 2010, pp. 240-315) describes the materials and methods used for the compositional analysis. In all, 75 different analytical components were meas ured, 68 in seed and seven in forage. Of the measured components, 26 in seed had more than 50% of the observations below the assay limit of quantitation (LOQ) and were excluded from the statistical analysis. The overall data set was examined for evidence of biologi cally relevant changes.

The compositional analyses confirmed that MON 87769 had the intended change in fatty acid composition, while the other components analyzed in MON 87769 were compositionally equivalent to conventional soybean. As in tended, MON 87769 seed contained relatively high

levels of SDA and GLA and low le vels of two minor fatty acids, trans-SDA and trans-ALA that were not found in conventional soybean (Mons anto 2010, Table VII-1, p. 101). The two trans fatty acids are known to form during seed extrac tion and processing. The mean percent of total fatty acid and mean percent dry weight for each of these fatty acids combined across sites is

summarized below for MON 87769.

Seed Fatty Acid (FA) % Total FA Range % Dry Weight Range 18:4 Stearidonic (SDA) 26.13 16.83 - 33.92 3.94 2.77 - 4.91

18:3 γ -Linolenic (GLA) 7.09 6.07 – 8.03 1.09 0.93 - 1.22 18:4 6c,9c,12c,15t (trans-SDA) 0.18 0.058 - 0.26 0.027 0.011 – 0.036 18:3 9c,12c,15t (trans-ALA) 0.44 0.38 – 0.48 0.068 0.055 – 0.081

Statistical analysis was conducted on the compos ition data for other components for evidence of biologically-relevant changes. 18: 2 LA is the most abundant fatty acid in conventional soybean. Considering LA is the starting material from which SDA and GLA are produced, the LA levels were significantly different (lower) in M ON 87769 (18.46 % of total FA) compared to conventional soybean A3525 (54.90 % of total FA). As anticipated, the LA values were also

well outside the 99% tolerance inte rval for the population of conve ntional references as well as the range of values found in the published literature and the Inte rnational Life Science Institute Crop Composition Database ( http://www.cropcompositi on.org/query/index.html ; Ridley et al. 2004). In addition to LA, combined -site statistical differences were found in five other fatty acid levels (16:0 palmitic, 18:1 oleic, 18:3 linolenic, 20:0 arachidic, and 22:0 behenic acid). The difference in the level of each of these five fatty acids was relatively small in absolute magnitude (5% or less of total fatty acids ) and/or their mean values a nd ranges in MON 87769 seed were

within the 99% tolerance interv al for the population of the co nventional reference varieties (Monsanto 2010, Table VII-2, pp. 106-112, and Ta ble E-4 pp. 260-261). Given the intended shift in the fatty acid metabolism toward an increase in SDA content in MON 87769, differences in fatty acid levels were expected.

Statistical comparisons between MON 87769 and th e conventional control for the presence of other components showed that 22 non-fatty acid analytes in soybean seed were significantly different (p<0.05) in the combined-site anal ysis (Monsanto 2010, Table VII-2, pp. 106-112 and Appendix Table E-14, pp. 294-295). For the 22 non-fatty acid analytes, most of the statistical differences between MON 87769 and conventional soybe an control were seen in amino acids (17 out of the 22 significant differen ces) and the magnitude of these differences were small (10% or less). The mean and range values of these ami no acids, as well as those of seed proteins,8 carbohydrates, and isoflavones which were also si gnificantly different in the combined site analysis, were within the calculated 99% tolera nce interval for the popu lation of commercial conventional soybean varieties and also within the range of values found in the published

literature and the International Life Sc ience Institute Crop Composition Database ( http://www.cropcomposition.org/query/index.html ; Ridley et al. 2004). The three isoflavones, genistein, daidzein and glycitein, were the only antinutritional f actors among those described as naturally occurring in soybean seed (which also include trypsin inhibitors, lectins, stachyose, raffinose, and phytic acid) that were found to be significantly different in the combined site analysis. These differences were not considered to be biologi cally meaningful from a food and feed safety or nutritional perspe ctive. Combined-site analysis of forage showed no significant differences (p>0.05) between MON 87769 a nd the conventional soybean control.

In addition to the compositional analysis of seed and forage, four soybean processed fractions (oil, meal, lecithi n, and protein isolate) were produced from MON 87769 seed and subjected to compositional analysis in a ccordance with OECD guidelines (OECD 2001). As expected, apart from the intended fatty acid ch ange, the composition of the soybean processed fractions from MON 87769 is equi valent to the composition of s oybean processed fractions from the conventional soybean control (Monsanto 2010, Appendix Table E-15 to E-18, pp. 303-313). Thus, the processed fractions from MON 87769 are c oncluded to be as safe and nutritious as the processed fractions prepared from conventiona l soybean. Collectively, the compositional data support the conclusion that MON 87769, with the excep tion of the expected changes in fatty acid composition, does not have biologically meaningful differences from conventional soybean from a food/feed safety and/or nutritional perspective.

Based on all the above noted considerations, APHIS concludes that MON 87769 poses no more of a plant pest risk from new gene products, changes to plant metabo lism or composition (with the exception of intended cha nges in fatty acid composition) than conventional soybean.



### Weediness of MON 87769
Soybean is a highly domesticated le gume species, and cultivated vari eties of soybean in the U.S. do not exhibit weedy characteristics, nor is s oybean listed as a weed in any major weed references (Crockett 1977; Holm et al. 1979; Muenscher 1980). Likewise, soybean is not

identified as a noxious weed in the Fe deral Noxious Weed List (7 CFR § 360; http://plants.usda.gov/java /noxious?rptType=Federal ). Moreover, soybean does not possess any of the attributes commonly associated with weed s (Baker 1965), such as long persistence of seed in the soil, the ability to disp erse, invade, and become a domina nt species in new or diverse landscapes, or the ability to compete well with native vegetation.

Phenotypic and agronomic characteristics of MON 87769 were evaluated in a comparative manner to assess plant pest poten tial (OECD 1993). These assessments included 14 plant growth and development characteristics, five seed germ ination parameters, two pollen characteristics, six plant-symbiont interaction characteristics, an d observations for plant-insect and plant-disease interactions and plant responses to abioti c stressor (Monsanto 2010, Table VIII-1, p. 116). Monsanto presented the following results to show that Mon 87769 is phenotypically and agronomically similar to conventiona l control of reference varieties:9  Seed dormancy is one of the potential traits effecting volunteerism and weediness. A total of 25 comparisons were made betw een MON 87769 and the conventional control for seed germination characteristics (Mons anto 2010, Tables VIII-1 and VIII-2, p. 121- 122). No statistically or bi ologically significant differences were detected between MON 87769 and the conventional control for percent normal germinated, abnormal germinated, viable hard, dead, or viable firm-swollen seed in the AOSA temperature regime (20/30 °C) or for percent germinated, viable hard, dead, or viable firm-swollen seed in the additional temperature regimes (10, 20, 30, 10/20, 10/30 °C; Monsanto 2010, Table VIII3. P. 122). Furthermore, the germination of both MON 87769 (89.1%) and the conventional control (92.1%) at 20/30 oC exce eded the accepted standard of 80% minimum germination for certified soybean seed recommended by the official seed certifying agencies of North America (AOSCA 2003). Thus, the germination values of

MON 87769 were not different from the A 3525 conventional control, an established commercial soybean variety, and were within the range of accepted germination values for certified soybean seed. Soybean lacks se ed dormancy (Monsanto Field Test Reports) and a majority of viable seeds germinate under adequate temperature and moisture. Although soybean seeds can poten tially grow as volunteer plan ts in a subsequent crop rotation, volunteer plants are most likely be killed by frost in the soybean growing regions during autumn or winter of the y ear they were produced. Even if soybean volunteers get established, there are effective weed management strategies to control such volunteers (OECD 2000; Yo rk et al. 2005).

 Fourteen plant growth, development, and yiel d characteristics (early stand count, seedling vigor, plant growth stage, days to 50% flowering, flower color, plant pubescence, plant height, lodging, pod shattering, fi nal stand count, seed mois ture, 100 seed weight, test weight, and yield.) were a ssessed under field conditions as part of the plant

characterization assessment of MON 87769. Da ta were collected at 21 field locations over two years that provided a diverse range of environmental and agronomic conditions representative of commercial soybean pr oduction areas in the U.S. (Monsanto 2010, Table VIII-4, p. 125). The results of this as sessment showed the in troduced trait did not unexpectedly alter the phenotypic or agronom ic characteristics of MON 87769 compared to conventional soybean and are indicativ e of no increased weediness of MON 87769 (Monsanto 2010, Tables VIII-5 and VIII-6, pp. 1 26-127; for a detailed field analysis see Monsanto 2010, Appendix G, pp. 321-340).

 Increased pollen viability a nd altered pollen morphology migh t be beneficial for plant species to acquire weedy characteristics su ch as higher seed production or increased pollen dispersal facilitating gene flow and invasiveness (Lu et al. 2008). Pollen was collected from MON 87769, the control (A3525) , and four commercial reference soybean varieties grown under similar agronomic c onditions. No statis tically significant

differences were detected be tween MON 87769 and the contro l for percent viable pollen or pollen grain diameter (Monsanto 2010, Tabl e VIII-7, p. 130). Furthermore, no visual differences in general pollen morphology were observed between MON 87769 and the

control. These results demonstrate that the introduced genes and proteins produced from them did not alter the overall morphology or viability of MON 87769 pollen compared to the conventional soybean control. Since soybean is highly self-pollinating, this10 observation is consistent with the similar seed yield obtained from MON87769 and the control (A3525) soybean in field tests.

 A total of 703 comparative observations of plant response to abiotic stressors, disease damage, and arthropod damage were conducted in field trials at 21 sites over two consecutive years to assess environmental interactions of MON 87769 compared to the conventional control (see Monsanto 2010, A ppendix I, pp. 344-364 for details). These

data were used to assess th e susceptibility and tolera nce of MON 87769 to specific abiotic stressors, diseases, or arthropod pe sts compared to that of the conventional soybean control. No biologically meaningful differences were observed across sites over two U.S. soybean growing seasons between MON 87769 and the conventional control in their susceptibility or tolera nce to specific abiotic stressors , diseases, or arthropod pests or in their interactions with specific pests.

Results of these evaluations indicate that there is no fundamental difference between MON

87769 and the conventional control for traits associ ated with weediness. Collectively, these findings support the conclusion th at MON 87769 is no more likely to be a weed compared to conventional soybean.



### Impacts on the Weediness of Any Other Plant with which MON 87769 can

Interbreed
Gene flow is a natural biological process with si gnificant evolutionary importance. A number of angiosperm taxa are believed to be derived fr om hybridization or introgression between closely related taxa (Grant 1981; Soltis and Soltis 1993; Rieseberg 1997; He gde et al. 2006), and even in the existing floras, the occurrence of hybridization or introgression is reported to be widespread (Stace 1987; Rieseberg and Wendel 1993; Peterson et al. 2002). It has been a common practice by plant breeders to artificially introgress traits from wild relatives into crop plants to develop new cultivars. However, gene flow from crops to wild relatives is also thought of as having a potential to enhance the weedine ss of wild relatives, as observed in rice, sorghum, sunflower and few other crops (see Table 1 in Ellstrand et al. 1999).

Soybean is a predominantly a self-pollinated species (OECD 2000), yet a small amount of outcrossing does occur in this species (Mons anto 2010, Table X-3, p. 178). A majority of outcrossing occurs within a couple of meters fr om the source populations and falls to about 0.01% or less within 10 – 15 meters (s ee Monsanto 2010, Table X-3 for outcrossing investigations). Based upon thes e factors, it is unlikely that MON 87769 will naturally outcross or hybridize to a signif icant extent with other soybean va rieties in agricultural settings.

In assessing the risk of gene introgression from MON 87769 into its sexually compatible relatives, APHIS considers two primary issues: 1) the potential for gene flow and introgression and, 2) the potential im pact of introgression.

The genus Glycine is divided into two subgenera, Glycine and S oja . The subgenus Soja consists of three annual species: G. soja Sieb. and Zucc., the wild form of soybean; G. gracilis Skvortz., the weedy form of soybean; and G. max, the cultivated soybean. These species grow wild or

semi-wild in Asia. Fertile hybrids between G. max and G. soja (Nakamaya and Yamaguchi11 2002; Mizuguti et al. 2010), and between G. max and G. gracilis (Karasawa 1952) occur. Glycine soja and G. gracilis grow naturally only in Asia, not in the United States (see Table 9.1 in Lu 2005). The subgenus Glycine consists of twelve wild pe rennial species. These species grow wild in Australia, South Pacific Islands and Asia (Newell et al. 1978), and do not exist naturally in the U.S. Hybrids between perennial Glycine species are fertile.

Glucine max is the only Glycine species located in the United Stat es, thus there are no other plant species with which G. max can interbreed. Glycine max has never been found in the wild (Hymowitz and Singh 1987) without human interventi on. Therefore, it is highly unlikely that soybean plants in the United States will be found outside of an agricultura l setting. It is also highly unlikely that gene flow and introgr ession will occur between MON 87769 and soybean plants in a natural environment. USDA has ther efore determined that an y adverse consequences of gene flow from MON 87769 to wi ld or weedy species in the Un ited States are highly unlikely.



### Effects of MON 87769 on Non-target Organisms
MON 87769 soybean is not engineered for pest resistance, thus ther e are no ‘target’ species, and thus no ‘nontarget’ species either. APHIS asse ssed whether exposure or consumption of MON 87769 soybeans would have an adverse affect on bene ficial species or wild life associated with soybeans. As discussed earlier, MON 87769 is sim ilar in nutritional and compositional analysis to nonmodified control soybean variety A3525 excep t for the intended changes in the fatty acid composition associated with the produc tion of SDA and GLA in the seed.

Monsanto also assessed the potential allerg enicity and toxicity of introduced traits ( Δ 15D and

Δ 6D proteins obtained from P. juliae and N. crassa , respectively) according to the

recommendations of the Codex Alimentarius Commission (Codex 2003). Donor organisms of the two genes are not known to be to xic, and one of the donor organisms, N

. crassa , is found in the digestive tracts of vertebrate species, incl uding humans. Monsanto’s bioinformatic analyses of both proteins demonstrated that they do not share structurally or immunologically relevant amino acid sequence similarities with known a llergens. Additionally, Monsanto conducted digestive fate experiments with the Δ 6D and Δ 15D proteins and found that the full-length proteins are rapidly digested in simulated gastric fluid (SGF), a characteristic shared among many proteins with a history of safe consumption. The transiently stable protein fragments in the SGF assay were quickly degraded during a short e xposure to simulated intestinal fluid (SIF). Rapid digestion of the full-length pr oteins in SGF and SIF, together with rapid degradation of the transiently stable fragments from the SGF assay by SIF, indicates that it is highly unlikely that

the Δ 6D and Δ 15D proteins and their fragments will r each absorptive cells of the intestinal mucosa. Finally, the Pj Δ 6D and Nc Δ 15D proteins are present at ve ry low concentrations which average no more than 0.00043% and 0.00239%, resp ectively, of the tota l protein in MON 87769

mature seed.

Monsanto also assessed both proteins for their po tential toxicity. Struct urally and functionally similar proteins (homologous desaturases) are pr esent across life forms and also found in human and animal diets. The proteins lack structural similarity to k nown toxins or bi ologically active proteins known to have adverse effects to mammals . Both proteins occur at extremely low levels in the harvested seed, in other words, they make up a negligib le portion of the total protein present in food and feed derived from MON 87769. Monsanto provided data showing that12 feeding high rates of the Pj Δ 6D and Nc Δ 15D proteins purified from MON87729 immature seed did not cause adverse effects on mice (Monsanto 2010, p. 98).

In addition, the changed fatty acid composition of mature MON 87769 soybean is not expected to adversely affect organisms th at might feed on them. The envi ronmental exposure and fate of fatty acids in MON87769 was analyzed in Sect ion X.A.3.2 . and X.A.3.3. of the Monsanto 2010, (pp. 169-171). Monsanto provided the results of a published 90 day/one generation reproductive rat toxicity study in which a no observable adverse effect level (NOAEL) of 4 g of SDA soybean oil/kg/day (the maximum dose tested) was dete rmined (Hammond et al., 2008) (see Monsanto 2010, Appendix F for experimental de tails, pp. 316-320). In practic al terms, given that fatty acids constitute 18% of the dry weight of M ON 87769 soybean seeds, and the 100 seed weight is 15.1 g (as reported in the Monsanto 2010,), this would constitute a consumption rate of approximately 66 seeds of MON 87769 soybean /day for a typical 500 g adult rat. The SDA and GLA are found in variety of s ources (fish, plant seeds, algae), and in mammals these are

intermediates in the in vivo metabolism of ALA to long chain om ega-3 fatty acids and of LA to arachidonic acid, respectively.

Furthermore, Monsanto has submitted food and feed safety data to FDA as a voluntary consultation process. Based on the food and feed safe ty data, lack of toxic ity and allergenicity of introduced gene products, APHIS concludes that feeding of MON 87769 plant or seed from mammals and other nontarget organisms is unlikely to cause any adverse impact on their survival and reproduction.

Beneficial arthropods, pollinators, symbiotic nitr ogen fixing bacteria, and wildlife associated with soybeans are unlikely to be adversely a ffected by cultivation of MON 87769 soybean. No significant reduction in bene ficial arthropods was detected in observations of field trials (see Monsanto 2010, Section VIII.D.2.2.). Neither benefi cial arthropods nor pollinators feed directly on soybean seed, and thus would not be expected to be affected. Bradyrhizobium japonicum are included in seed inoculums to improve nitrogen fixation in the field. The Monsanto included studies (summarized in Monsanto 2010, S ection VIII.D.4. on pp.130-133, Tables VIII-9 and VIII-10, and in Appendix K) that demonstrate that MON 87769 soybean was not altered in its symbiotic relationship with B. japonicum compared to conventional soybean based on assessments of nodule number, shoot nitrogen, an d biomass of nodules, shoot material, or root

material.



### Transfer of Genetic Information to Organisms with which MON 87769 cannot

Interbreed
The horizontal gene transfer (HGT ) between unrelated organisms is one of the most intensively studied fields since 1940, and the issue gained ex tra attention with the release of transgenic plants into the environment (Dröge et al. 1998). HGT has been im plicated as a major contributor to the spread of antibiotic resistance amongst pa thogenic bacteria and the emergence of increased virulence in bacteria, eukaryot es, and viruses; and has contri buted to major transitions in evolution. Gene exchange has been documente d for nearly all types of genes and between unrelated organisms (Gogarten et al. 2002). For example, r ecently, Yoshida and colleagues (Yoshida et al. 2010) through a co mparative genomics analysis implicated HGT for the presence13 of a similar genetic sequence between the parasitic plant purple witchweed ( Striga hermonthica ), which infests cereal fields (monocots), and sorghum ( Sorghum bicolor ).

APHIS examined the potential for the new gene tic material inserted into MON 87769 to be horizontally transferred to other organisms w ithout sexual reproduction and whether such an event could lead directly or indi rectly to disease, damage, injury or harm to plants. The MON 87769 contains two noncoding sequences from bacteria . Horizontal gene tr ansfer and expression of DNA from a plant species to other bacterial species is unlikely to occur based on the following observations.

Although there are many opportunities for plants to directly interact wi th fungi, bacteria, and parasitic plants (e.g. as commensals, symbionts, pa rasites, pathogens, decomposers, or in the guts of herbivores), so far there are no reports of si gnificant horizontal gene transfer between sexually incompatible or evolutionarily distant organi sms (as reviewed in Keese 2008). Accumulated evidence show that there are universal gene-tra nsfer barriers, regardless of whether transfer occurs among closely or distan tly related organisms (Kaneko et al. 2000; Koonin et al. 2001; Wood et al. 2001; Kaneko et al. 2002; Brown 2003; Sorek et al. 2007). Many genomes (or parts thereof) have been sequenced fr om bacteria that ar e closely associated with plants including Agrobacterium and Rhizobium (Kaneko et al. 2000; Kaneko et al. 2002; Wood et al. 2001). There is no evidence that these organisms contai n genes derived from plants. In cases where review of sequence data implied that horizontal gene transfer o ccurred, these events are inferred to occur on an evolutionary time scale on the order of millions of years (Koonin et al. 2001; Brown 2003), so also the case with the recent re port about of HGT between sorghum and purple witchweed. According to authors (Yoshida et al. 2010), the incorporati on of a specific genetic sequence occurred between sorghum and purpl e witchweed before speciation of purple witchweed ( S. hermonthica ) and related cowpea witchweed ( S. gesnerioides ), a parasitic plant of dicots, from their common ancestor. In other words, HGT is an extremely rare event, and a majority of those rare events occur over millions of years.

Transgene DNA promoters and coding sequences are optimized for plant expression, not prokaryotic bacterial expression. Thus even if horizontal gene transf er occurred, proteins corresponding to the transgenes are not likely to be produced. FDA has evaluated horizontal gene transfer from the use of antibiotic resistance marker genes, and concluded that the

likelihood of transfer of antibioti c resistance genes from plant ge nomes to microorganisms in the gastrointestinal tract of humans or animals, or in the environment, is remote (FDA 1998: http://www.fda.gov/Food/GuidanceComplianceRe gulatoryInformation/GuidanceDocuments/Biot echnology/ucm096135.htm ). Therefore APHIS conc ludes that horizontal gene transfer is unlikely to occur from Mon 87769 to microorganisms and thus no significant plant pest risk is expected from horizontal gene transfer.



### Conclusion
APHIS has reviewed the information submitted by th e petitioner and conducted a plant pest risk assessment on MON 87769 soybean. Due to the lack of plant pest risk from the inserted genetic material, the lack of atypical res ponses to disease or plant pests in the field, the lack of weediness characteristics of MON 87769 soybean, the lack of deleterious eff ects on non-targets or14 beneficial organisms in the agro-ecosystem, and the lack of horizontal gene transfer, APHIS concludes that MON 87769 soybean is unlik ely to pose a plant pest risk.15 References AOSCA (Association of Official Seed Cer tifying Agencies). 2003. "Yellow Books" 2003 Operational Procedures, Crop Standards and Se rvice Programs Publica tion (Genetic and Crop Standards). Association of Official Seed Certifying Agencies, Meridian, Idaho.

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