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# Draft **Plant Pest Risk Assessment**  **for Double**  **Herbicide -** **Tolerant Soy**  **bean (**  **G lycine**  **max**

**) event**  **FG72 Petition**  **09 -**  **328 -**  **01p September**  **2011 United**  **States Department of**  **Agriculture Ma**  **rketing and Regulatory**  **Programs Animal and**  **Plant Health Inspection**  **Service**

## Ta **ble of Content s** 

Table of Contents ................................ ................................ ................................ ...................... 2 Plant Pest Risk Assessment of Bayer CropScience Event FG72 Soy bean ............................... 3 A. Introduction ................................ ................................ ................................ ............. 3 B. History

of Development of Event FG72 Soy bean ................................ .................. 4 C. Description of the Modifications ................................ ................................ ............ 6 D. Plant Pest Risk Assessment ................................ ................................ .................... 7 E. Conclusion ................................ ................................ ................................ ............ 14 F. References ................................ ................................ ................................ ............. 14

## Plant **Pest Risk As**  **sessment of Bayer CropScience** 

**Ev ent FG72 Soybean**  **A. Intr** **oduction** Baye r CropScience (BCS) has petitioned APHIS for a determination that event FG72 double herbicide - tolerant soy

bean is unlikely

to pose a plant pest risk ( BCS, 2009 ) and, therefore, should no longer be a regulated article under APHIS’ 7 Code of Federal Regulations (CFR) part 340. AP HI S administers 7 CFR part 340 under the authority of the plant pest provisions o f the Plant Protection Act of 2 001 1 . This plant pest risk assessment was conducted to determine if FG72 so ybean is unlikely to pose a plant pest risk. The event FG72 so ybean ( Glycine max cultivar Jack) expres s es two added proteins which impart herbicide tolerance , 2mEPSPS from corn (Zea mays ) and 4 - hydroxy phenylpy ruvate dioxygenase (HPPD), from Pseudomonas fluorescens (strain A32) . The 2mEPSPS and HPPD protein s expressed in the soy bean protect the plant from the application of the herbicides gl yphosate and isoxaflutole (IFT), respectivel y. FG72 so ybean was produced b y transformation of soybean tissue using direct gene transfer (biolistics) . Because two of the regulatory sequenc es used to facilitate expression of the herbicide tolerance genes in FG72 soy bean ( Tobacco Etch Virus promoter and A. tumefaciens nos terminator) were derived from plant pests, the FG72 soy bean has been considered a regulated article under APHIS regulation s at 7 CFR part 340. Potential impacts to be addressed in this risk assessment are those that pertain to the use of FG72 soy bean and its progeny i n the absence of confinement. APHI S uses data and information submitted by

the applicant, in addition to current literature, to determine if FG72 so ybean 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 th at organism. Potential impacts in this Plant Pe st Risk Assessment are those that pertain to plant pest risk characteristics. APHIS regulation 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 interbreed. An anal ys is of agricultural or cultivation practices associated with FG72 soy bean and impacts on the environment are considered in the Environmental Assess ment (EA) for FG72 so ybean . A thorough assessment of the effects of the determination on nontarget organisms, beneficial organisms and threatened and endangered species will also be considered in the EA.

1 Section 403 (14) of the Plant Protection Act (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, 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.”4



### B. **History**

**of Development of Event FG72 Soy**  **bean** The f irst gly phosate tolerant soy

bean that was given a determination of nonregulated status in May , 1994 by

APHI S was Roundup Ready® soy bean 40 - 3 - 2 (OECD Unique Identifier MON 04 032 - 6; submitted by Monsanto in p etition number 93 - 258 - 01p ) . This event involved so ybean that contained the DNA as follows: an enhanced 35S promoter from cauliflower mosaic virus, a chloroplast transit peptide from Petunia hybrida fused to the 5 - enolp yruv ylshikimate - 3 - phosphate sy

nthase (EPSPS) gene from Agrobacterium s pp. strain CP4 , an d the nopaline sy nthase 3’ terminator from A. tumefaciens . Monsanto completed its food safety consultation with the U.S. Food and Drug Administration (FDA) on soy bean Event 40 - 3 - 2 on January 27, 1995 (http://www.accessdata.fda.gov ; BNF 0000 1 ). In Septemb er 1993, the Environmental Protection Agency

(EPA) completed its Reregistration E ligibility D ecision (RED) on the pesticide active ingredient gl yphosate ( EPA - 738 - F - 93 - 011 ). In 1995 , the Plant Biosafety Office (PBO) of the Canadian Food Inspection Agency ( CFIA) concluded that Monsanto’s Event 40 - 3 - 1 soy bean was substantially

equivalent to currently

grown soy

bean , in terms of their potential environmental impact and livestock feed safety and the novel traits would not have any substantial negative effect on the environment (CFIA Decision Document DD 95 - 05 ; http://www.inspection.gc.ca). In 2006, Monsanto developed a second generation gl yphosate tolerant soy

bean called RReady2YieldTM, Event MON 89788. APHI S made a det ermination of nonregulated status for Event 89788 (RReady2YieldTM) on July 23, 2007. EPA and FDA did not re - evaluate MON 89788 because the gene of interest was identical to Event 40 - 3 - 2. MON 89788 is very similar to MON - 04032 - 6. Both plants were genetical ly engineered to be gl yphosate tolerant by

inserting a gene (from Agrobacterium spp . strain CP4) coding for epsps into the soy be an genome. The three major differences between MON 89788 and MON - 04032 - 6 are: the use of a different promoter for the cp4 epsps gene, a different transformation method , and the use of a different recipient variety . The Food Standards Australia New Zealand (FSANZ) approved MON 89788 soy

bean in a pre - market safety assessment on August 8, 2007 (Application A592) for import (but not fo r cultivation). There are no historical genetically engineered events for the insertion of the hppd gene into soy bean or any other crop species. Page 33 of the BCS submitted petition describes the action of the protein made b y the hppd gene along with di agram for the two metabolic pathway s in plants; the biosy nthesis of plastoquinone and bio sy nthesis of tocopherol:

“ The biochemical pathway s in which HPPD is involved differ between plants and nonphotosy

nthetic organisms. In bacteria and animals, it merely serves catabolic purposes b y catalyzing the first committed step in ty rosine degradation that in the end yi elds energetically exploitable glucogenic and ketogenic products ( Brownlee, Johnson Winters et al., 2004 ) . In plants, however, it is also involved in several anabolic pathway s; its reaction product homogentisate (2,5 - dihydroxyohenylacetate) being the aromatic precursor of tocopherol and plastoquinone, which are essential to the photosy nthetic transport chain and antioxidative systems ( Fritze, 2004 ) . ” **Figure 1. Biochemical pathways of HPP**  **D proteins**5

a) catabolism of ty

rosine; b) biosy

nthesis of plastoquinone (plants) ; c) biosy nthesis of tocopherol (plants) The first herbicide developed in this family was isoxaflutole by

Rhone Poulenc (now Aventis) and was registered for use in corn in 19 9 8 by the EPA (FR Vol. 63, No.104 50773) . From the EPA Isoxaflutole Fact Sheet ( EPA, 1998 ) : “Isoxaflutole is a pigment inhibitor. It works b y preventing the biosynthesis of carotenoid pigments, which protect chlorophyl l from decomposition by sunlight. Without carotenoid pigments, chlorophyl l pigments are photo - oxidized and chloroplasts break down. Without the energy collecting action of the chlorophyl l, the whole plant eventuall y dies.” Once in the plant, isoxaflutole is rapidly converted into diketonitrile (DKN) which works b y inhibiting the production o f 4 - hydrox yphenyl - py ruvate dioxygenase (HPPD). Inhibition of HPPD stops the biosy

nthesis of plastoquinone (Figure 1) . Plastoquinone is a carotenoid pigment that is needed for electron transport process of photosy nthesis (http://www.uniprot.org/key words/618 ). HPPD controls a broad spectrum of grass and broadleaf weeds and is effective on difficult to control weeds such as velvetleaf and wooly cupgrass (Z ollinger, 2009 ) . HPPD is called a proherbicide because it degrades rapidl y to the active molecule diketonitrile . The primary6

breakdown product is mobile and breaks down more slowly than isoxaflutole itself ( Zollinger, 2009 ) . DKN is reactivated by rainfall events, providing control of small weeds that have emerged. The combined effects of desorption (release of the compound from the soil) and degradation resupply

the soil solution with a bioactive product . Because of its adsorption characteristics , the application rate of isoxaflutole is adjusted for soil texture and organic matter. Depending on the concentration of o rganic matter , the half - life of HPPD in soil can be up to 30 day s ( Inoue, Oliveira et al., 2009 ) . Because of the potential for leaching, especiall y on permeable soil s, isoxaflutole is a restricted - use pesticide. The label prohibits use o n sandy soils with less than 2% organic matter where the water table is less than 25 feet from the surface. I soxaflutole has a bleaching effect as chlorophyl l is broken down in sunlight but is not replaced. The sy mptoms first appear on leaf edges and tip s because this is the site of new carotenoid sy nthesis ( Zollinger, 2009 ) . Since the FG72 so yb ean is tolerant to isoxaflutole, this herbicide can be applied prior to planting (pre - plant) without any

signif icant damage to the plant while being toxic to weeds not managed by gl yphosate. Baye r CropScience has now developed the transgenic soy bean , Event FG72, which express es both 2mEPSPS protein and HPPD proteins . Event FG72 so ybean has been developed by BCS as an alternative soy

bean product that could help manage herbicide resistant weed populations . Event FG72 so ybean has been field tested under APHI S regulations since 200 1 . Data were provided in the petition for field trials completed prior to the petition submission. Field test reports can be found in the BCS Event FG72 soybean petition ( BCS, 2009, pp. 93 ) . BCS will make a request to the Environmental Protection Agency (EPA) to allow the use of an isoxaflutole formulation to be used on Event FG72 soy bean. BCS initiated the consultation process with FDA for the commercial distribution of FG72 so ybean and submitted a safety and nutritional assessment of food and feed derived from FG72 so ybean to the FDA on December 3, 2009. FDA is currentl y evaluating the submission, and as of March 16, 2012, has not completed the consultation. 

### C. **Description of the M**  **odification s**  Event FG72 so ybean contains the stably integrated genes 2m epsps and hppdPfW 336, which encode respectively

the 2mEPSPS and HPPD proteins. The 2mepsps and the hppdPfW336 genes were introduced into the soy bean genome b y means of direct gene transfer (biolistics) . “ Event FG72 contains a single insert of two consecutive complete copies of the linear fragment. ” ( BCS, 2009, pp.25 ) . BCS’ double herbicide - tolerant soy bean (Event FG72) has been genetically engineered to contain the following transgene fragments: The 2mepsps gene expression c assette borne b y pSF10 is represented b y the following string: “Ph4a748 - intron1 h3At - TPotpC:: 2mepsps ::3’histonAt” ( BCS, 2009, pp.22 ) :7

**Ph4a748 promoter and intron 1 h3At:** The Ph4a748 promoter sequence is derived from the histone H4 gene of Arabidopsis thaliana ( Chabouté, Clement et al., 1987 ) and controls expression of the 2mepsps gene. The Ph4a748 promoter, combined with the first intron of gene II of the histone H3.III variant of Arabidopsis thaliana ( Chaubet, Clement et al., 1992 ) directs high level constitutive expression, especiall y i n rapidly growing plant tissues. **TPotp C transit peptide:** The optimized transit peptide, which contains sequences from the RuBisCO small subunit genes of corn and sunflower, targets the mature protein to the plastids, which is where the wildtype protein would be located ( Lebrun, L eroux et al., 1996 ) . **2mepsps coding sequence:**  The wildtype epsps gene isolated from maize (Zea mays ) was mutated using site - directed mutagenesis. Two point mutations resulted in the double mutant 2mepsps gene ( Lebrun, Sailland et al., 1997 ) . A methionine codon is added to the N - terminal of the 2mEPSPS protein sequence in order to restore the cleavage site of the optimized plastid transit peptide. The 2mepsps gene encodes a 47 kDa protein consisting of 445 amino acids. **3’histonAt terminator:** The 3’ untranslated region of the histone from Arabidopsis thaliana ( Chabouté, Clement et al., 1987 ) . The hppdPfW336 gene expression cassette is represented by the following string: “Ph4a748 ABBC - 5’tev - TPotpY:: hppdPfW 336 ::3’nos” (p.23, BCS Event FG72 petition: **Ph4a748 ABBC promoter and 5’tev enhancer:** The same Ph4a748 promoter was used to drive the expression of the hppdPfW 336 gene, but an internal portion of the promoter sequence (referred to as “B”) was duplicated to increase the promoter activity in plant cells. In combination with the leader sequence of the tobacco etch virus ( 5' tev; Carrington and Freed, 1990 ) , this promoter brings the level of exp ression of hppdPfW336 gene to an appropriate level that enables tolerance at agronomic doses of IFT. **TPotp Y transit peptide:** The optimized transit peptide, which contains sequences from the RuBisCO small subunit genes of corn and sunflower, targets the mature protein to the plastids, which is where the wildtype protein would be located ( Lebrun, L eroux et al., 1996 ) . **hppdPfW336 gene coding sequence:**  The wildty pe hppd gene isolated from Pseudomonas fluorescens was mutated using site directed mutagenesis. A point mutation resulted in the hppdPfW 336 gene ( Boudec, Rodgers et al., 2001 ) . The hppdPfW 336 gene encodes a 40 kDa protein consisting of 358 amino acids. **3' nos terminator :**  The 3’ untranslated region of the nopaline sy nthase from Agrobacterium tumefaciens is a polyadenylation signal ( Depicker, Stachel et al., 1982 ) . 

### D. **Plant Pest Risk Assessment**8

This plant pest risk assessment is to determine whether BCS Event FG72 is unlikely to pose a plant pest risk. If APHIS determines that a GE organism is unlikely to pose a plant pest risk, APHI S then has no regulatory authority over that organism under 7 CFR part 340. APHI S administers the regulations 7 CFR part 340 under the authority of the Plant Protection Act of 2000 (PPA). The Animal and Plant Health Inspection Service of the United States Department of Agriculture (USDA - APHIS) has prepared a Plant Pest Risk Assessment in response to a petition (APHI S Number 09 - 328 - 01p) from BCS . APHI S regulation 7 CFR 340.6(c) stipulates the information needed for consideration in a petition for nonregulated status. APHI S evaluate d information submitted by

the applicant related to plant pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new enzy mes, or changes to plant meta bolism, potential impacts on non - target organisms, weediness of the regulated article, and any impacts on the weediness of any other plant with which it can interbreed. Issues related to agricultural or cultivation practices are considered in the Environm ental Assessment for BCS Event FG72 soy bean . Based on information on the biology of soy bean ( OECD, 2000 ) , data presented by BCS ( BCS, 2009, Appendix I , pp. 93 - 114 ) and scientific data relevant to a discussion of plant pest risk, APHI S concluded the following regarding BCS double herbicide - tolerant soy bean Event FG72 : 

### Potential Impacts of Genetic Modifications on Disease and Pest Susceptibilities USDA - APHIS assessed whether BCS Event FG72 soy bean is likely

to have significantly increased disease and pest susceptibility . The assessment encompasse s a thorough con sideration of introduced traits , their impact on agronomic traits and plant composition, and interactions with pests and diseases. BCS observed field trials for damage due to diseases (Alternaria leaf spot, Anthracnose, Asian rust, Bacterial blight, Bacterial pustule, Brown spot, Brown 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 insect pests ( a phid, b ean leaf beetle, b lister beetle, c orn rootworm, f

lea beetle, grasshopper, green cloverworm, Japanese beetle, l eafhopper, l eafroller, Mexican bean beetle, s pidermite, s eed corn maggot, s oy bean stem borer, s tink bug, t arnished plant bug, thistle caterpillar, thrips, v elvetbean caterpillar, white fl y, w ireworm, and w ool ybear caterpillar) for four consecutive ye ars conducted across different agroecological conditions (from 2001 - 2004) and then for thre e ye ars (2007 - 2009) . Data were collected on pest and disease damage across 1 5 sites from 2001 - 2003 and 1 8 sites from 2007 - 2009 . The observations submitted by BCS indicated no meaningful differences between Event FG72 soy bean and the non - transgenic counter

parts for diseases or insect pests ( BCS, 2009, Section VII.D, Table 22 ) . Thus Event FG72 is expected to be susceptible to the same plant pathogens and insec t pests as conventional soy

bean with the noted exceptions listed in the variety registration for Jack. From the BCS petition (pp. 65) “ The variety registration of Jack, claims resistance to soybean cyst nematode (SCN) (Races 3 and 4) Heterodera glycines Ichinohe) and susceptibility to9

Phytophthora rot (Races 1, 4, and 7) caused b y Phytophthora megasperma (Drechs.) f. sp. glycinea T. Kuan & D.C. Erwin. Neither of these phy topathologies was observed consistentl y in the trials, so it was not possible to confirm xpression of these variety traits. At the Perry location, one pl ant of event FG72 was presumed to have died of Phytophthora root rot. ” BCS’ aforementioned data indicate that Event FG72 soy bean is not biologically

different from conventional soy

bean (with the exception of the HPPD and 2mEPSPS proteins) and the herbicid e resistant phenoty pes did not alter the pest and disease incidences on Event FG72 soy bean ; therefore, Event FG72 soy bean is no more susceptible to pests and diseases compared to conventional soy

bean cultivars. 

### Expression of the Gene Product, New Enzymes **, or Changes to Plant Metabolism** The HPPD and 2mEPSPS protein s expressed in Event FG72 so ybean are nearl y identical to the native protein s produced by Pseudomonas fluorescens and corn, respectively . The HPPD protein was modified b y the replacement of the amino acid gl ycine with a try ptophan and the 2mEPSPS protein has two point mutations along with a methionine codon added to the N - terminal end. The functional activities of the HPPD and 2mEPSPS proteins were confirmed in vivo from field grown Event FG72 soy

bean plants ( BCS, 2009, pp. 38 ) . BCS collected samples from grain, seeds, leaf samples from three different growth stages (V4, V6, V8), stem and root at V4 and V8 stages to quantify HPPD and 2mEPSPS protein expression in Event FG72 soy bean ( BCS, 2009 , Appendix 2 ) . Both proteins were expressed at varying levels during all stages of the plant life cycle ( BCS, 2009, Table 7 and 8 ) . USDA - APHIS assessed whether changes in plant metabolism or composition in Event FG72 soy bean is likely

to alter plant pest risk. The assessment encompasses a consideration of the expressed added protein s or enzy mes and their effect on plant metabolism and an evaluation of whether the nutrients and anti - nutrient levels in harvested seed and forage derived from Event FG72 so ybean are comparable to those in the non - transgenic parent line , Jack variety as well as three commercial so ybean varieties , Stine 2686 - 6, 2788 and 3000 - 0. ( BCS, 2009, Tables 25 - 29, pp. 71 - 74 ) . Detailed composi tional and nutritional comparisons of Event FG72 so yb ean and the conventional soy

bean control , Jack , were conducted on samples collected from ten sites across the U.S. in 200 8 and compared against reference ranges calculated from three commercial soy bean v arieties listed in the above paragraph . The anal ys is included moisture, protein, fat, ash, carbohydrates, amino acids, fatty acids, anti - nutrients and isoflavones , consistent with OECD guidelines ( OECD, 2001 ) . Appendix 2.G ( BCS, 2009, pp. 134 ) describes the materials and methods used for the compositional analy sis. The overall data set was examined for evidence of biologicall y relevant changes. All Event FG72 soy bean compositional anal ys is values were similar to the two test and control entries and fell within the commercial variety and literature reference ranges ( BCS, 2009, Tables 25 - 29, pp. 71 - 74 ) . The compositional anal yses confirmed that Event FG72 soy bean is compositionally

equivalent to the three conventional soy bean lines (Stine 2686 - 6, 2788 and10

3 000 - 0) with no single data entry outside any reference ranges (commercial varieties or literature references) . Collectivel y, the compositional data support the conclusion that Event FG72 soy bean does not have biologicall y 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 Event FG72 soy bean poses no more of a plant pest risk from new gene products, changes to plant metabolism or compositi on (with the exception of intended changes in herbicide resistance ) than conventional soy bean. 

### Weediness of **Event FG72 Soybean** Soy bean is a highl y domesticated legume species, and cultivate d varieties of soy bean in the US do not exhibit weedy

characteristics, nor is soy bean listed as a weed in any major weed references ( Muenscher, 1952 ; Crockett, 1977 ; Holm, Pan cho et al., 1979 ) . Likewise, so ybean 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, soy 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. Phen oty pic and agronomic characteristics of Event FG72 soy bean were evaluated in a comparative manner to assess plant pest potential ( OECD, 1993 ) . These assessments included 17 plant growth and development characteristics: early stand count, plant vigor, day s to flowering, flower color, leaf shape, health rating at stage V4 - 5 , health rating at stage R1, health rating of mature plants, pubescence color, pod color, hilum color, canop y, days to maturity, yi eld in bu/ac, lodging, final stand count and po d shatter ( BCS, 2009, Table 57, pp. 181 ) . BCS presented the following results to show that Event FG72 so ybean is phenoty pically and agronomicall y similar to conventional control reference varieties:

 Seed dormancy

is one of the potential traits effecting volunteerism and weediness. No statistically

or biologic ally

significant differences were detected between Event FG72 soy bean and the conventional control ( BCS, 2009, Table 24, pp. 64 ) . Seed tests were completed b y Iowa State Seed Lab using standard testing ( BCS, 2009, pp. 133 ) . No dormant s eeds were identified. There was a slight difference (94% vs. 96%) in germination at day

6 ( BCS, 2009, Table 24, pp. 64 ) between Event FG72 and the parent line Jack, but when germination was extended to 13 day s, the percent viable seed of the samples (95% vs. 96%) were statisticall y similar. The germination values of Event FG72 soy bean were not different from the conventional control and were within the range of accepted germination values for certified so ybean seed. Although soybean seed s can potentially

grow as volunteer plants in a subsequent crop rotation, volunteer plants would most likely

be killed by

frost in the soy

bean growing regions during autumn or winter of the year they

were produced. Even if soy bean volunteers get establish ed, there are effective weed management strategies to control such volunteers ( OECD, 2000 ; York, Beam et al., 2005 ) .11

 The results of the 17 plant growth and development characteristics showed the introduced trait did not unexpectedly alter the phenotypic or agronomic characteristics of Event FG72 so ybean compared to conventional soy bean . The early

plant density

and plant vigor ratings were lower in unsprayed Event FG72 plots than in commercial variety plots which carried through to late season possibly due to seed lot quality . The day s to 50% flowering were shorter for Event FG72 compared to commercial varieties, but for day s to 95% flowering, there were no differences between the Event FG72 so yb ean and commercial varieties. There are indicative of no increased weediness of Event FG72 soy bean ( BCS, 2009, pp. 53 - 6 8 ) . Results of these evaluations indicate that there is no fundamental difference between Event FG72 soy bean and the conventional control for traits associated with weediness. Collectively , these findings support the conclusion that Event FG72 soy bean is no more likel y to be a weed compared to conventional soy bean. 

### Impacts on the Weediness of Any Other Plant with which **Event FG72 Soybean can Interbreed**  Gene flow is a natural biological process with significant evolutionary importance. A number of ang iosperm taxa are believed to be derived from hy bridization or introgression between closel y related taxa ( Grant, 1991 ; Rieseberg and Wendel, 1993 ; Soltis and Soltis, 1993 ; Hegde, Nason 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, Pearman 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 weediness of wild relatives, as observed in rice, sorghum, sunflower and few other crops ( see Table 1 in Ellstrand, Prentice et al., 1999 ) . Soy bean is predominantly

a self - pollinated species ( OECD, 2000 ) , yet a small amount of outcrossing does occur. Soy bean typically exhibits a level of cross - pollination below one p ercent. Adjacent rows are measured to have between 0.03 and 3.62% outcrossing, but plants more than 4.5 meters apart cross at less than 0.02% ( Caviness, 1966 ; Yoshimura, Matsuo et al., 2006 ) . While most sources agree that insects do not greatly increase outcrossing rate in domestic soy bean ( Erickson, 1984 ) , there is some evidence that some insects (notably honey bees) can increase hybridization rates ( Free, 1970 ; McGregor, 1976 ) . Pollen is only viable for 2 - 4 hours (it desiccate s quickly) with anthesis (pollen shed) normally occurring in the late morning ( Caviness, 1966 ) . Current cultivation practices to prevent out - crossing have been deemed sufficient to prevent unwanted gene flow. For so ybean, the Association of Official Seed Certifying Agencies (AOSCA) mandates a zero isolation distance where “ Fields of so ybeans shall be separated from any other variety or uncertified seed of the same variety b y a distance adequate to prevent mechanical mixture ” . Based upon these factors, it is unlikely that Event FG72 soy

bean will naturally

outcross or hy bridize to a significant extent with other soy bean varieties in agricultural settings.12

In assessing the risk of gene introgress ion from Event FG72 so ybean 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 consists 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. These species grow wild or semi - wild in Asia. Fertile hy brids between G. max and G. soja (Nakayama and Yamaguchi, 2002 ; Mizuguti, Ohigashi et al., 2010 ) , and b etween G. max and G. gracilis ( Karasawa, 1952 ) occur. Glycine soja and G. gracilis grow naturally onl y in Asia, not in the United States ( Lu , 2005 ) . The subgenus G lycine consists of twelve wild perennial species. These species grow wild in Australia, South Pacific Islands and Asia (Newell and Hy mowitz, 1978 ) , and do not exist naturally in the U S. Hy

brids between perennial Glycine species are fertile. Gl ycine 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 ( Hy mowitz and Singh, 1987 ) without human intervention. Therefore, it is highl y unlikely that soy bean plants in the United States will be found outside of an agri cultural setting. It is also highl y unlikely that gene flow and introgression will occur between Event FG72 soy bean and soy bean plants in a natural environment. USDA has therefore determined that any adverse consequences of gene flow from Event FG72 soy b ean to wild or weedy

species in the United States are highl y unlikely. 

### Effects of **Event FG72 Soybean on Non**  **- target Organisms**  Event FG72 so ybean is not engineered for insect pest resistance, thus there are no ‘target’ species, and thus no ‘nontarget’ speci es either. APHI S assessed whether exposure or consumption of Event FG72 soy beans would have an adverse effect on beneficial species or wildlife associated with soy beans. As discussed earlier, Event FG72 soy bean is similar in nutritional and compositional anal ys is to unmodified control so ybean variety except for the intended changes in herbicide resistance associated with the production of HPPD and 2mEPSPS proteins in the plant . BCS also assessed the potential allergenicity and toxicity of introduced trait s ( HPPD and 2mEPSPS proteins obtained from Pseudomonas fluorescens and corn , respectivel y) according to the recommendations of the Codex Alimentarius Commission ( Codex, 2003 ) . The donor organism of the 2mepsps gene (corn) is not toxic and consumed b y humans and animals alike and the donor organism of the hppd gene, Pseudomonas fluorescens , can be an opportunistic pathogen in immunocompromised patients , but is generall y seen as having poor virulence due to its inability

to multiply

at body temperature and having to compete against host defenses ( Liu, 1964 ) . BCS ’s bioinformatic analys es of both proteins demonstrated that they do not share structurally

or immunologically

relevant amino acid sequence similarities with known allergens. Additionall y, BCS conducted digestive fate experiments with the HPPD and 2mEPSPS proteins and found that the full - length proteins are rapidl y digested in simulated gastric fluid (SGF), a characteristic13

shared among many proteins with a history of safe consumption. The transiently stable protein fragments in the SGF assay were quickl y degraded during a shor t exposure to simulated intestinal fluid (SIF). Rapid digestion of the full - length proteins in SGF and SIF, together with rapid degradation of the transiently stable fragments from the SGF assay by SIF, indicates that it is highly unlikel y that the HPPD and 2mEPSPS proteins and their fragments will reach absorptive cells of the intestinal mucosa. Finall y, the HPPD and 2mEPSPS proteins are present at very lo w concentrations which average no more than 0.00 023% and 0.041 %, respectively , of the total protein in Event FG72 soy bean mature seed. BCS also assessed both proteins for their potential toxicity . HPPD is ubiquitous in nature across all kingdoms including bacteria, fungi, plants and animals. HPPD has been characterized in organisms present in human food such as carrot, barley , pork and beef ( BCS, 2009, pp. 38 ) . The proteins 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 ( see above paragraph for concentrations) , in other words, they make up a negligible portion of the total protein present in food and feed derived from Event FG72 so yb ean . BCS provided data showing that feeding high rates of the HPPD and 2mEPSPS proteins purified from Event FG72 soy bean immature seed did not cause adverse effects on mice ( BCS, 2009, pp. 47 - 52 ) . Furthermore, BCS has submitted food and feed safety data to FDA as part of 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 Event FG72 soy bean plant or seed by mammals and other nontarget organisms is unlikely to cause any adverse impact on their survival and reproduction. 

### Tr **ansfer of Genetic Information to Organisms with which Event FG72 Soybean**  **cannot Interbreed** 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 th e release of transgenic plants into the environment ( Dröge, Puhler 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, Doo little et al., 2002 ) . For example, recently , Yoshida and colleagues (Yoshida, Maruyama et al., 2010 ) through a comparative genomics analysis 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 Event FG72 soy bean to be horizontally

transferred to other organisms without sexual reproduction and whether such an event could lead directl y or indirectl y to disease, damage, injury or harm to plants. The Event FG72 soy bean contains one coding sequence from bacteria, the modified hppdPfW 336 gene from Pseudomonas fluorescens ( as described b y Boudec, Rodgers et al., 2001 ) and two non - coding regulatory

sequences from the bacteria, Agrobacterium tumefaciens . Horizontal gene transfer14

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, parasites, pathogens, decomposers, or in the guts of herbivores), so far there are no reports of significant horizontal gene transfer b etween 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 distantl y related organisms ( Kaneko, Nakamura et al., 2000 ; Koonin, Makarova et al., 2001 ; Wood, Setubal et al., 2001 ; Kaneko, Nakamura et al., 2002 ; Brown, 2003 ; Sorek, Zhu et al., 2007 ) . Many genomes (or parts thereof) from bacteria that are closel y associated with plants have been sequenced including Agrobacterium and Rhizobium ( Kaneko, Nakamura et al., 2000 ; Wood, Setubal et al., 2001 ; Kaneko, Nakamura et al., 2002 ) . 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, Makarova et al., 2001 ; Brown, 2003 ) . This is simi lar to the case in a recent report about of HGT between sorghum and purple witchweed. According to the authors (Yoshida, Maruyama 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 w itchweed (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 did occur, 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 ) . Therefore APHI S concludes that horizontal gene transfer is unlikely to occur from Event FG72 soy bean to microorganisms and thus no significant plant pest risk is expected from horizontal gene transfer. 

### E. **Conclus ion**  APHI S has reviewed and conducted a plant pest risk assessment on BCS Event FG72 so ybean . Due to the lack of plant pest risk from the inserted genetic material, the lack of weediness characteristics of BCS Event FG72 so ybean , the lack of atypical respon ses to disease or plant pests in the field, 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 BCS Event FG72 so ybean is unlikely to pose a plant pest risk. 

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