1



## PLANT PEST RISK ASSESSMENT FOR **MON 87769 SOYBEAN**  Monsanto Company (Monsanto) has petition ed APHI S for a determination that the geneticall y engineered (GE) stearidonic acid soy bean ( Glycine max) event MON 87 769 **(**hereafter referred to as MON 87769 **)** is unli kely to pose a plant pest risk (Monsanto 2009) and, therefore, is no longer a regulated article under the APHIS ’ 7 Code of Federal Regulations ( CFR) part 340. APHI S administers 7 CFR part 340 under the authority of the plant pest provisions of the Plant P rotection Act of 2000 1

. This plant pest risk assessment was conducted to determine if MON 87769 is unlikely

to pose a greater plant pest risk than the unmodified soy bean line with which it was derived . MON 87769 was produced by transformation of soy

bean 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., tm l terminator sequence and left and right T - DNA border sequen ces from A. tumefaciens ) or eliminated (35S RNA from figwort mosaic virus) ; therefore, the engineered soy bean has been considered a regulated article under APHIS regulations at 7 CFR part 340. Potential impacts to be addressed in this risk assessment are those that pertain to the use of MON 87 769 and its progeny in the absence of confinement. APHI S uses data and information submitted by

the applicant, in addition to current literature, to assess if MON 87 769 is unlikely to pose a plant pest risk. If APHI S determines that a GE organism is unlikely to pose a plant pest risk, then APHI S has no regulatory authority over that organism. Potential impacts in this Plant Pest Risk Assessment are those that pertain to plant pest risk characteristics. APHIS regulati on 7 CFR 340.6(c) specifies the information needed for consideration in a petition for nonregulated status. APHI S will evaluate information submitted by the applicant related to plant pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new enz ym es, 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 information to organisms with which it cannot inter breed. An anal ys is of agricultural or cultivation practices and indirect plant pest effects 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 MON 87769 . Likewise, a thorough assessment of the effects of the determination on nontarget organisms , beneficial organisms , and threatened and endangered species will be considered in the EA.

1 Plant Protection Act in 7 U.S.C. 7702 § 403(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, cause 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 fungus. (F) A virus or viroid. (G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding subparagraphs.”2 

## Introduction **a. History of Developm**  **ent of MON 87769**  **Stea ridonic Acid**  **(SDA) Soybean**  Soy bean 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. (Hy mowitz and Singh 1992 ). Monsanto developed SDA MON 87769 by u sing recombinant DNA technology to geneticall y engineer (GE) into a conventional soy bean variety two foreign desaturase genes, Primula juliae Δ 6 desaturase ( Pj. D6D ) and Neurospora crassa Δ 15 desatura se ( Nc. F ad3) , whose combined action result s in the production of two fatty acids, stearidonic acid (SDA) and γ - linolenic acid (G LA), that do not occur in conventional soy

beans ( Figure 1 below, adapted from Figure VII - 1 , p. 103, Monsanto 2010 ). Δ 9 Desaturase Δ 12 Desaturase Δ 15 Desaturase 18:0 18:1 18:2 18:3 stearic acid oleic acid linoleic acid α - linolenic acid (LA) (ALA) Δ 6 Δ 6 Desaturase Desaturase

Δ 15 Desaturase 18:3 18:4 γ - l inolenic stearidonic acid acid (GLA) (SDA) Pathway s where soy

bean endogenous desaturases work Pathway s where exogenous (engineered) desaturases work **Figure 1. Fatty acid biosynthesis in plants and the introduced changes to changes to produce MON 87769**  **Soybean (ada pted from** **Monsanto 2010, Figure VII**  **- 1**  **)** MON 87769 seed oil consists of approximately 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 om ega - 3 fatty acids (Harris et al. 2008) . SDA is an omega - 3 poly unsaturated fatty acid (PUFA) composed of an 18 - carbon chain with four double bonds (18:4) . Although SDA is found in fish, fish oils, green leafy vegetables, and some nuts and vegetable oils , it occurs relativel y high levels in only a limited number of fish, and seaweed, and rarely in other plant species used as food sources ( Figure 2 in Guil - Guerrero 2007 ; Whelan 2009 ) . In mammals, SDA is a metabolic intermediate in the production of the long er carbon 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 (p. 4, Monsanto 2010 ) . Although the therapeutic and health - promoting effects of consuming omega - 3 long chain PUFAs from fish (SA, EPA, and DHA) are widel y recognized, ty

pical Western diets contain very little fish, and the same benefits may not be shared b y 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 ty

pical Western diet (Whelan 2009) . However, the lower number of double bon ds 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 alter native to fish oil in food products. While 6% of the world’s fish oil is used for human con sumption and an even amount for animal feed, 87 % is used in fish feed largel y t o support aquaculture (Whelan 2009) . O ils 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 feed efficiency in some fish species (e.g. Arctic char , cod, Atlantic salmon , and hy brid 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 om ega - 3 fatty acids in food and animal feed (poultry and aquaculture) . 

### b. **Description of the**  **Inserted Genetic**  **Elem ents**  MON 87769 was developed through A . tumefaciens mediated transformation of soy bean line A3525, a nontransgenic , mid - maturity group III, co nventional variety with very high yield potential (Steffen 2004). The disarmed A . tumefaciens used for the transformation harbors a plasmid vector PV - ZMP Q1972 that contains two separate T - DNAs (Figure IV - 1, p. 45 , Monsanto 2010 ) . The first T - DNA, designated as T - DNA I, contains two expression cassettes: Primula juliae Δ 6 desaturase ( Pj.D 6 D ) gene expression cassette and Neurospora crassa Δ 15 desaturase 3 (

Nc.F ad3) gene expression cassette. The second T - DNA region (T - DNA II) contains the cp4 epsps gene exp ression cassette that encodes the CP4 EPSPS protein (5 - enolpy ruvylshikimate - 3 - phosphate sy nthase protein from Agrobacterium sp. strain CP4) that provides tolerance to the action of gly phosate, which is the active ingredient in Roundup® agricultural herbici des. T - DNA I region incorporated into MON 87769 contain ed the following genetic elements (Table V - 1, p. 50 , Monsanto 2010 ) :  Border Sequence: Left and right T - DNA border sequences from A . tumefaciens to facilitate T - DNA transfer ( Depicker et al. 1982; B ar ker et al. 1983).  7S α Promoter : Promoter and leader from the Sphas1 gene of so ybean encoding beta congl ycinin storage protein (Doyle et al. 1986) that directs mRNA transcription in seed.  Δ6D 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 carbox ylend of a fatty acid .  tm l terminator: 3 ′ untranslated region of the tml gene from A . tumefaciens octopine - ty pe Ti plasmid (Kemp et al. 2000) .4

 7Sα promo ter: Promoter and leader from the Sphas2 gene from so ybean encoding the alpha subunit of beta - congl ycinin storage protein (Wang and Dubois 2004) that directs mRNA transcription in seed.  F ad (fatty acid desaturase) 3 gene: F ad3 gene coding sequence from Neu rospora crassa (Ursin et al. 2006) that codes for Δ 15 desaturase , which creates a double bond at the 15 th position from the carboxyl end of a fatty acid .  E- 9 terminator: 3 ′ untranslated region of the pea ribulose 1,5 - bisphosphate carbox yl ase small subunit 2 ( rbcS2) gene which functions to direct pol yadenyl ation of the mRNA (Coruzzi et al. 1984). In addition to the above - mentioned genetic el ements, the insert also contain ed non coding intervening sequences of 8 - 101 base - pair length that were used for DNA cloning. T - DNA II region, **which was bred out of the final MO**  **N 87669 event** , contained the follo wing genetic sequences ( Figure IV - 1, pp . 45 - 47 , Monsanto 2010 ):  Border Sequence: Left and right T - DNA border sequences from A. tumefaciens to facilitate T - DNA transfer (Depicker et al. 1982; Bar ker et al. 1983).  FMV promoter: Promoter for the 35S RNA from figwort mosaic virus (FMV) (Rogers, 2000) that directs transcription in plant cells.  ShkG leader sequence: 5'untranslated 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 transport of the CP4 EPSPS protein to the chloroplast.  cp4 epsps coding sequence: Codon modified coding s equence 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 b y removing the pathogenic sequences encoding phyt ohormones that cause crown gall tumors from the Ti (tumor inducing) plasmid present in the Agrobacterium (Koncz and Schell 1986);  the final product does not contain any of the backbone sequences outside of the T - DNA I borders from the transformation vector, PV - GMPQ1972 (Figure s V - 3, p. 53 ; V - 5, p. 59 , Mo nsanto 2010 );  the final product does not contain T - DNA II elements that contain the cp4 epsps gene (Figure V - 6, p. 62 , Monsanto 2010 );  the DNA inserted into the soy bean genome is present at a single locus and contains one functional cop y of D 6

D ( Figure V - 8 , p. 69 , Monsanto 2010 ) and fad3 (Figure V - 11, p. 72 , Monsanto 2010 ) 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 sequen ce alignment between the MON 87769 insert sequence to the PV -5

GMPQ1972 transformation vector sequence) ( Figure V - 3, p.53 and p. 81 , Monsanto 2010 ) ;  no novel open reading frames were created that spa nned either the 5 ′ or 3′ junctions between the T - DNA and soy bean genomic sequences;  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 (Figures V - 14 and V 15, pp. 78 - 79 , Monsanto 2010 ) from five segregating generations tested (Monsanto 2010, Figure V - 13, p. 77) in the breeding history . The stability was further confirmed b y the Mendelian inheritance of the T - DNA I in MON 87769 (Table V - 3, p. 84 , Monsanto 2010 ) in three segregating generations (Figure V - 17, p. 83 , Monsanto 2010 ). Because the T - DNA II region , containing the herbicide 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 plant pest risk assessment discussion presented in the following paragraph s .



## PLANT PEST RISK ASSESSMENT Based on information on the biology of so ybean (OECD 2000), data presented by Monsanto Company ( Monsanto 2010 ), and scientific data relevant to the discussion of plant pest risk, APHI S makes the following conclusions for MON 87769: 

### Potential Im

**pacts of Genetic Modifications on Disease and Pest Susceptibilities** USDA - APHIS assessed whether MON 87769 so ybean is likely to have significantly

increased disease and pest susceptibility . This assessment encompasses a thorough consideration of introduced traits, their impact on agronomic traits and plant composition, and interactions with pests and diseases. Mons anto used well - established qualitative or quantitative techniques (Appendix I , pp. 344 - 346 , Monsanto 2010 ) to measure field trials for damage due to diseases (Alternaria leaf spot, Anthracnose, Asian rust, Bacterial blight, Bacterial pustule, Brown spot, B rown stem rot, Cercospora leaf blight, Charcoal rot, Downy mildew, Frogeye leaf spot, Fusarium , Phytophthora , Powdery

mildew, Pythium , Rhizoctonia , Sclerotinia , Septoria , Soy bean mosaic virus, Soy

bean rust, Stem canker, Sudden death, White mold) and insec t pests (Aphid, Bean leaf beetle, Blister beetle, Corn rootworm, Flea beetle, Grasshopper, Green cloverworm, Japanese beetle, Leafhopper, Leafroller, Mexican bean beetle, Spidermite, Seed corn maggot, So ybean stem borer, Stink bug, Tarnished plant bug, This tle caterpillar, Thrips, Velvetbean caterpillar, White fly, Wireworm, and Wooly bear caterpillar) for two years conducted across different agroecological conditions. Data were collected on pest and disease damage across 17 sites in 2006 and 4 site s in 2007 , and 4 sites in 2006 and 2007 were quantitatively tested for abundance of arthropods. There was a slight difference in susceptibility 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 collection point and for leafhopper and stinkbug at one collection point at one location (MI). However, these differences were either within the range of the reference varieties and /or were not found to be significantly different in other locations. The data submitted by Monsanto indicated no meaningful differences between6

MON 87769 soy

bean and the non - transgenic counterparts for diseases (Appendix Tables I - 3 and I - 4, pp. 349 - 350 , Monsanto 2010 ), or insect pests (Appendix Tables I - 5 to I - 7, pp. 351 - 355; Table I - 9, pp. 358 - 360 , Monsanto 2010 ). Thus MON 87769 is expected to be susceptible to the same plant pathogens and insect pests as conventional soy bean. Monsanto’s aforementioned data (expression of the gene products, nutritional and compositional anal ys es) indicate that MON 87769 is not biologically different from conventional soy bean (with the exception of the Δ6D and Δ 15D proteins) and modified fatty acid phenoty

pes did not alter the pest and disease incidences on Mon 87769; therefore, MON 87769 is no more susceptible to pests and diseases compared to conventional so yb ean cultivars. 

### Expression of **the Gene Pro duct, New Enzym**  **es, or Changes to Plant Metabolism** The Δ 6D protein expressed in MON 87769 is nearly identical to the native protein produced b y Primula juliae containing 446 amino acids and three histidine motifs that are characteristic of integral membran e desaturases (Figure VI - 1, pp. 87 - 89 , Monsanto 2010 ; Ursin et al. 2008 ) . Furthermore, the petitioner’s analy sis of the Δ 6D deduced amino acid sequence has shown that, the MON 87769 Δ 6D is similar to other Δ 6 desaturases in amino acid composition and enzy m atic structure (Nakamura and Nara 2004) . Likewise, the Δ15 D 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 alanine 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 ve ctor. The MON 87769 - expressed Δ 15D protein consists of 429 amino acids and three histidine motifs that are ch aracteristic of integral membrane desaturases (Figure VI - 3. p. 89 , Monsanto 2010 ). Th e functional activities of the Δ6D and Δ15D proteins were confirmed in vivo after expression from the yeast expression vector in S accha romyces cerevisiae (see pp. 87 - 90 , Monsanto 2010 for details) . Monsanto collected tissue samples from leaf, forage, root, mature seed and immature seed to quantify

Δ6 D and Δ 15D protein expression in MON 87769 (see Appendix D for the protein expression anal ys is, pp. 237 - 239 , Monsanto 2010 ) . Because the expression of the PjΔ6D and NcΔ15D proteins is driven by a 7Sα' and 7Sα seed specific promoter, respectivel y , those two proteins were not detected in leaf and root tissues of MON 87769, while b oth proteins were detected in immature seed and at much lower levels in mature seed and forage , which contains a small of amount of residual immature seeds (Tables VI - 1, p. 95 and VI - 2, p. 96 , Monsanto 2010 ). The highest level s of PjΔ6D and NcΔ15D proteins measured in immature seeds of MON87769 were 210 and 330 μg/g of tissue on a dry weight (DW) basis , respectivel y , and in the mature seed they

represent no more than 0.00043% and 0.00239% of the total protein . As expected, the Δ6D and Δ 15D proteins were not detected in the conventional soy bean control, A3525 (p. 94 , Monsanto 2010 ). USDA - APHIS assessed whether changes in plant metabolism or composition in MON 877 69 is likely to alter plant pest risk. The assessment encompasses a consideration of the expressed protein or enzy

mes and their effec t on plant metabolism and an evaluation of whether the nutrients and antinutrient levels in harvested seed and forage derived from MON 87769 are comparable to those in the conventional so ybean control, A3525.7

Detailed compositional and nutriti onal comparisons of M ON 87769, the conventional soy bean control A3525 , and ten commercially available soy

bean varieties were conducted on samples collected from fives sites across the U.S. in 2006 . The anal ys is included protein, fat, carbohydrates, fiber, ash, moisture, amino acids, fatty acids, vitamin E, and antinutrients and key secondary metabolites, consistent with OECD guidelines (OECD 2001 ) . Appendix E (pp. 240 - 315 , Monsanto 2010 ) describes the materials and methods used for the compositional analysis. In all, 75 diff

erent anal yt ical components were measured, 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 analy sis. The overall data set was examined for evidence of biologicall y relevant changes. The compositional anal yses confirmed that MON 87769 had the intended change in fatty acid composition, while the other components anal yz ed in MON 87769 were compositionally equivalent to conventional soy bean. As intended, MON 87769 seed contained relativel y high levels of SDA and GLA and low levels of two minor fatty acids, trans - SDA and trans - ALA that were not found in conventional soy bean ( Table VII - 1, p. 101 , Monsanto 2010 ) . The two trans fatty acids are known to form during s eed extraction 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 877 69 . **Seed Fatty Acid**  **(FA) %**  **Total F A**  **Range %**  **D ry**  **W eight**  **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) 0.4 4 0.38

– 0.48 0.068 0.055

– 0.081 Statistical analy sis was conducted on the composition data for other components for evidence of biologicall y- relevant changes. 18:2 LA is the most abundant fatty acid in conventional soybean. Considering LA is the st arting material from which SDA and GLA are produced, the LA levels were significantly

different (lower) in MON 87769 (18.46 % of total FA) compared to conventional soy

bean A3525 (54.90 % of total FA) . As anticipated, the LA values were also well outside the 99% tolerance interval for the population of conventional references as well as the range of values found in the published literature and the International Life Science Institute Crop Composition Database (http://www.cropcomposition.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 relativel y small in absolute magnitude ( 5% or less of total fatty

acids) and/or their mean values and ranges in MON 87769 seed were within the 99% tolerance interval for the population of the conventional reference varieties ( Table VII - 2, pp. 106 - 112 , and Table E - 4 pp. 260 - 261 , Monsanto 2010 ) . Given the intended shift in the fatty

acid metabolism toward an increase in SDA content in MON 87769, differences in fatty acid level s were expected. Statistical comparisons between MON 87769 and the conventional control for the presence of other components showed that 22 non - fatty acid analytes in so ybean seed were significantly different (p<0.05) in the combined - site anal ys is ( Table VII - 2, pp. 106 - 112 and Appen dix Table8

E - 14, pp. 294 - 295 , Monsanto 2010 ) . For the 22 non - fatty acid anal yt es, most of the statistical differences between MON 87769 and conventional soy bean control were seen in amino acids (17 out of the 22 significant diff erences) and the magnitude of these differences were small (10% or less ). The mean and range values of these amino acids , as well as those of seed proteins , carbohydrates , and isoflavones which were also significantly different in the combined site anal ysis, were within the calculated 99% tolerance interval for the population of commercial conventional soy

bean varieties and also within the range of values found in the published literature and the International Life Science Institute Crop Composition Datab ase (http://www.cropcomposition.org/query /index.html ; Ridley

et al. 2004) . The three isoflavones , genistein, daidzein and gl yc itein, were the only antinutritional factors among those describe d as naturally occurring in soy

bean seed (which also include try psin inhibitors, lectins, stachyose, raffinose, and phytic acid) that were found to be significantly different in the combined site anal ys is. These differences were not considered to be biolo gically meaningful from a food and feed safety or nutritional perspective. Combined - site anal ys is of forage showed no significant differences (p>0.05) between MON 87769 and the conventional soy bean control. In addition to the compositional analy sis of se ed and forage, four so ybean processed fractions (oil, meal, lecithin, and protein isolate) were produced from MON 87769 seed and subjected to compositional analy sis in accordance with OECD guidelines (OECD 2001) . As expected, apart from the intended fatty acid change, the composition of the soy bean processed fractions from MON 87769 is equivalent to the composition of soy bean processed fractions from the conventional so ybean control (Appendix Table E - 15 to E - 18, pp. 303 - 313 , Monsanto 2010 ) . Thus, the proce ssed fractions from MON 87769 are concluded to be as safe and nutritious as the processed fractions prepared from conventional soy bean. Collectivel y, the compositional data support the conclusion that MON 87769, with the exception of the expected changes in fatty acid composition, does not have biologically meaningful differences from conventional soy bean from a food/feed safety

and/or nutritional perspective. Based on all the above noted considerations, APHI S concludes that MON 87769 poses no more of a p lant pest risk from new gene products, changes to plant metabolism or composition than conventional soy

bean. The expression of the introduced Δ 6 desaturase and Δ15 desaturase proteins and intended changes in fatty acid composition do not appear to affect pest or disease susceptibility . 

### Weediness of MON 87769 Soy bean is a highl y domesticated legume species, and cultivated varieties of soy bean in the U.S. do not exhibit weedy characteristics , nor is soy bean listed as a weed in any m ajor weed references (Crockett 1977; Holm et al. 1979; Muenscher 1980) . Likewise, soy bean is not identified as a noxious weed in the Federal Noxious Weed List (7 CFR § 360 ; http://plants.usda.gov/java/noxious?rptTy pe=Federal ). Moreover, s oy bean does not possess any of the attributes commonly associated with weeds (Baker 1965), such as long persistence 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 vegetation. Phenoty pic and agronomic characteristics of MON 87769 were evaluated in a comparati ve manner to assess plant pest potential (OECD 1993). These assessments included 14 plant growth9

and development characteristics, five seed germination parameters, two pollen characteristics, six plant - sy

mbiont interaction characteristics, and observation s for plant - insect and plant - disease interactions and plant responses to abiotic stressor (Table VIII - 1, p. 116 , Monsanto 2010 ) . Monsanto presented the following results to show that Mon 87769 is phenoty picall y and agronomicall y similar to conventional co ntrol of reference varieties:

 Seed dormancy

is one of the potential trait s effecting volunteerism and weediness . A total of 25 comparisons were made between MON 87769 and the conventional control for seed germination characteristics ( Tables VIII - 1 and VI II - 2, p. 121 - 122 , Monsanto 2010 ) . No statistically

or biologicall y 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 ( Association of Official Seed Anal ys ts ) 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 ; Table VIII - 3. p . 122 , Monsanto 2010 ) . Furthermore, the germination of both MON 87769 (89.1%) and the conventional control (92.1%) at 20/30 oC exceeded the accepted standard of 80% minimum germination for certified so ybean seed recommended by the Official Seed Certifying A gencies of North Am erica (AOSCA 2003). Thus, the germination values of MON 87769 were not different from the A3525 conventional control, an established commercial so ybean variety, and were within the range of accepted germination values for certified so ybean seed. Soy bean lacks seed dormancy (Monsanto Field Test Reports) and a majority of viable seeds germinate under adequate temperature and moisture. Although so ybean seeds can potentially grow as volunteer plants in a subsequent crop rotation, volunteer plants are most li kely be killed by frost in the soy bean growing regions during autumn or winter of the year they were produced. Even if so ybean volunteers get established, there are effective weed ma nagement strategies to control such volunteers (OECD 2000; York et al. 20 05).

 Fourteen p lant growth, development, and yi eld characteristics ( earl y stand count, seedling vigor, plant growth stage, day s to 50% flowering, flower color, plant pubescence, plant height, lodging, pod shattering, final stand count, seed moisture, 10 0 seed weight, test weight, and yi eld. ) were assessed under field conditions as part of the plant characterization assessment of MON 87769. Data were collected at 21 field locations over two years that provided a diverse range of environmental and agronom ic conditions representative of commercial soy bean production areas in the U.S. (Table VIII - 4 , p. 125 , Monsanto 2010 ). The results of this assessment showed the introduced trait did not unexpectedly

alter the phenoty

pic or agronomic characteristics of MON 87769 compared to conventional soy

bean and are indicative of no increased weediness of MON 87769 (Table s VIII - 5 and VIII - 6 , pp . 126 - 127 ; Monsanto 2010; f or a detailed field anal ys is see Appendix G, pp. 321 - 340 , Monsanto 2010 ) .  In creased pollen viability and altered pollen morphology might be beneficial for plant species to acquire weedy characteristics such as high er seed production or increased pollen dispersal facilitating gene flow and invasiveness (Lu et al. 2008). Pollen was collected from MON 8776 9, the control (A3525), and four commercial reference so yb ean varieties grown under similar agronomic conditions . No statistically significant10

dif ferences were detected between MON 87769 and the control for percent viable pollen or pollen grain diameter ( Table VIII - 7 , p. 130 , Monsanto 2010 ). Furthermore, no visual differences in general pollen morphology were observed between MON 87769 and the control. The se results demonstrate that the introduced genes and proteins produced from them did not alter the o verall morphology or viability

of MON 87769 pollen compared to the conventional so ybean control. Since soy bean is highl y self

- pollinating, this observation is consistent with the similar seed yield obtained from MON87769 and the control (A3525) so ybean 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 ye ars to assess environmental interactions of MON 87769 comp ared to the conventional control (see Appendix I , pp. 344 - 364 , Monsanto 2010 for details). These data were used to assess the susceptibility and tolerance of MON 87769 to specific abiotic stressors, diseases, or arthropod pests compared to that of the conventional soy bean control. No biologically meaningful differences were observed across sites over two U.S. soy

bean growing seasons between MON 87769 and the conventional control in their susceptibility

or tolerance to specific abiotic stressors, diseases, or arthropod pests or in their interactions with specific pest s . Results of these evaluations indicate that there is no fundamental difference between MON 87769 and the conventional control for traits associated with weediness. Collectively , these findings support the conclusion that MON 87769 is no more likely to be a weed compared to conventional soy

bean. 

### Im **pacts on the Weediness of Any Other Plant with which**  **MON 87769 can**  **Interbreed** Gene flow is a natural biological process with significant evolutionary importance. A number of angiosperm taxa are believed to be derived from hy bridization or introgression between closel y related taxa (Grant 1981; Soltis and Soltis 1993; Rieseberg 1997; Hegde et al. 2006), and even in the existing floras, the occur rence of hyb ridization or introgression is reported to be widespread ( S tace 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 d evelop new cultivars. However, gene flow from crops to wild relatives is also thought of as having a potential to enhance the weediness of wild relatives, as observed in rice, sorghum, sunflower and few other crops (see Table 1 in Ellstrand et al. 1999). Soy bean is a predominantly

a self - pollinated species (OECD 2000) , yet a small amount of outcrossing does occur in this species (Table X - 3, p. 178 , Monsanto 2010 ) . A majority of outcrossing occurs within a couple of meters from the source populations and falls to about 0.01% or less within 10

– 15 meters (see Table X - 3 , Monsanto 2010 for outcrossing investigations). Based upon these factors, it is unlikely that MON 87769 will naturally outcross or hy bridize to a significant extent with other soybean vari eties in agricultural settings.11

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 impact of introgression. The genus Glycine is divided into two subgenera, Glycine and S oja . The subgenus Soja con sists of three annual species: G. soja Sieb. and Zucc., the wild form of so ybean ; G. gracilis Skvortz., the weedy form of so ybean ; and G. max, the cultivated soy bean. The se species grow wild or semi - wild in Asia. Fertile hy brids between G. max and G. soja (Nakamaya and Yamaguchi 2002; Mizuguti et al. 2010 ), and between G. max and G. gracilis (Karasawa 1952) occur. Glycine soja and G. gracilis grow naturally onl y i n Asia, not in the United States ( see Table 9.1 in Lu 2005 ). The subgenus Glycine consists of twelve wild perennial species. These species grow wild in Australia, South Pacific Islands and Asia (Newell et al. 1978), and do not exist naturally in th e U.S. Hybrids between perennial Glycine species are fertile . Glycine max is the only Glycine species located in the United States, thus there are no other plant species with which G. max can interbreed. Glycine max has never been found in the wild (H ym owitz and Singh 1987) without human intervention. Therefore, it is highl y unlikel y that soy bean plants in the United States will be found outside of an agricultural setting. It is also highl y unlikely that gene flow and introgression will occur between MON 87769 and soy bean plants in a natural environment. USDA has therefore determined that any adverse consequences of gene flow from MON 87769 to wild or weedy s pecies in the United States are highl y unlikely. 

### Effects of **MON 87769 on Non**  **- target**  **Organism s**  MON 87769 soy

bean is not engineered for pest resistance, thus there are no ‘target’ species, and thus no ‘nontarget’ species either . APHIS assessed whether exposure or consumption of MON 877 69 soy

beans would have an adverse affect on beneficial species or wildlife associated with soy beans. As discussed earlier, MON 87 7 69 is similar in nutri tio nal and compositional analysis to nonmodified control soy bean variety A3525 except for the intended changes in the fatty acid composition associated with the production of SDA and GLA in the seed . Monsanto also assessed the potential allerg enicity and toxicity of introduced traits ( Δ6 D and Δ 15 D proteins obtained from P. juliae and N. crassa , respectivel y) according to the recommendations of the Codex Alimentarius Commission (Codex 2003). Donor organisms of the two genes are not known to be toxic, and one of the donor organisms, N . c rassa , is found in the digestive tracts of vertebrate species, including humans. Monsanto’s b ioinformatic anal ys es of both proteins demonstrated that they do not share structurally

or immunologicall y relevant amino acid sequence similarities with known al lergens. Additionally , Monsanto conducted diges tive fate experiments with the Δ6D and Δ 15D proteins and found that the full - length proteins are rapidl y digested in simulated gastric fluid (SGF), a characteristic shared among many proteins with a history

of safe consumption. The transientl y stable prot ein fragments in the SGF assay

were quickly

degraded during a short exposure to simulated intestinal fluid (SIF). Rapid digestion of the full - length protein s in SGF and SIF, together with rapid degradation of the transiently stable fragment s from the SGF assay by SIF, indicates that it is highl y unlikely that the Δ 6D and Δ 15D protein s and their fragment s will reach absorptive cells of the intestinal mucosa. Finally , the Pj Δ 6D and Nc Δ 15D protein s are present at very low concentration s which12

average no more than 0.00043% and 0.00239%, respectivel y, of the total pro tein in MON 87769 mature seed. Monsanto also assessed both proteins for their potential toxicity . Structurally and functionally similar proteins (homolog ou s desaturases) are present across life forms and also found in human and animal diets. The protein s lack structural similarity to known toxins or biologicall y 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 negligible portion of the total protein present in food and feed derived from MON 87769. Monsanto provided data showing that feeding high rates of the PjΔ 6D and NcΔ 15D proteins purified from MON87729 immature seed did not cause adverse effects on mice (p . 98 , Monsanto 2010 ). In addition, th e changed fatty acid composition of mature MON 87769 soybean is not expected to adversel y affect organisms that might feed on them. The environmental exposure and fate of fatty acids in MON87769 was anal yz ed in Section 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 soy bean oil/kg/day

(the maximum dose tested) was determined (Hammond et al. 2008) (see Appendix F for experimental details, pp. 316 - 320 , Monsanto 2010 ) . In practical terms, given that fatty acids constitute 18% of the dry weight of MON 87769 soy bean 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 soy bean /day for a typical 500 g adult rat. The SDA and GLA are found in variety of sources (fish, plant seeds, algae), and in mammals these are intermediates in the in vivo metabolism of ALA to long chain omega - 3 fatty acids and of LA to arachidonic acid, respectivel y. Furthermore, Monsanto has submitted food and feed safety data to FDA as a voluntary consultation process. Based on the food and feed safety data, lack of toxicity and allergenicity of introduced gene products, APHI S 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 arthrop ods, pollinators, sy mbiotic nitrogen fixing bacteria , and wildlife associated with soy beans are unlikely

to be adversely

affected by

cultivation of MON 87769 soy bean. No significant reduction in beneficial arthropods was detected in observations of field trials (see Section VIII.D.2.2 , Monsanto 2010 ). Neither beneficial arthropods nor pollinators feed directl y on soy bean seed, and thus would not be expected to be affected. Bradyrhizobium japonicum are included in seed inoculums to improve nitrogen fixati on in the field . The Monsanto included studies (summarized in Section VIII.D.4. on pp.130 - 133, Tables VIII - 9 and VIII - 10, and in Appendix K , Monsanto 2010 ) that demonstrate that MON 87769 soy bean was not altered in its sy mbiotic relationship with B. japon icum compared to conventional soy bean based on assessments of nodule number, shoot nitrogen, and biomass of nodules, shoot material, or root material. 

### Transfer of Genetic I

**nform ation**  **to Organism s with which MON 87769 cannot**  **I nterbreed** 13

The horizontal gene transfer (HGT) between unrelated organisms is one of the most intensively studied fields since 1940, and the issue gained extra attention with the release of transgenic plants into the environment (Dröge et al. 1998). HGT has been implicated as a major contributor to the spread of antibiotic resistance amongst pathogenic bacteria and the emergence of increased virulence in bacteria, eukaryotes, and viruses; and has contributed to major transitions in evolution. Gene exchange has been documented for nearl y all ty pes of genes and between unrelated organisms (Gogarten et al. 2002). For example, recently , Yoshida and colleagues (Yoshida et al. 2010) through a comparative genomics analy sis implicated HGT for the presence of a similar genetic sequence between the parasitic plant purple witchweed ( Striga hermonthica ), which infests cereal fields (monocots), and sorghum (Sorghum bicolor). APHI S examined the potential for the new genetic material inserted into MON 87769 to be horizontally

transferred to other o rganisms without sexual reproduction and whether such an event could lead directl y or indirectl y to disease, damage, injury or harm to plants . The MON 87769 contains two noncoding sequences from bacteria. Horizontal gene transfer 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 with fungi, bacteria, and parasitic plants (e.g. as commensals, sy mbionts, para sites, pathogens, decomposers, or in the guts of herbivores), so far there are no reports of significant horizontal gene transfer between sexually incompatible or evolutionarily distant organisms (as reviewed in Keese 2008). Accumulated evidence show that there are universal gene - transfer barriers, regardless of whether transfer occurs among closely

or distantly

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 (o r parts thereof) have been sequenced from bacteria that are closel y 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 contain genes derived from plants. In cases where review of sequence data implied that horizontal gene transfer occurred, 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 th e recent report about of HGT between sorghum and purple witchweed. According to authors (Yoshida et al. 2010), the incorporation of a specific genetic sequence occurred between sorghum and purple 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 transfer 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 antibiotic resistance genes from plant genomes to microorganisms in the gastrointestinal tract of humans or animals, or in the environment, is remote (FDA 1998 : http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/Biot echnology

/ucm096135.htm ). Therefore APHI S concludes that horizontal gene transfer is14

unlikely to occur from Mon 87769 to microorganisms and thus no significant p lant pest risk is expected from horizontal gene transfer. 

### Conclusion APHI S has reviewed the information submitted by the petitioner and conducted a plant pest risk assessment on MON 87769 soy bean . Due to the lack of plant pest risk from the inserted genetic material, the lack of atyp ical responses to disease or plant pests in the field, the lack of weediness characteristics of MON 87769 soy bean , the lack of deleterious effects on non - targets or beneficial organisms in the agro - ecos ys tem, and the lack of horizontal gene transfer, APHIS concludes that MON 87769 soy bean is unlikely

to pose a greater plant pest risk than the unmodified soy

bean line from which it was derived.15

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