## Plant Pest Risk Assessment for DAS
- 444Ø6

- 6 Soy b ean

**Table of Contents**

A. Introduction

................................ ................................ ................................ ............................... 2 B. Development of DAS

- 444Ø6

- 6 herbicide tolerant soybean

................................ ..................... 3 C. Description of the modification

................................ ................................ ................................ . 6 D. Potential for DAS

- 444Ø6

- 6 to have altered disease and pest susceptibilities

.......................... 8 E. Potential for effect on non

- target organisms, including those beneficial to agriculture

......... 10 F. Potential for enhanced weediness or invasiveness

................................ ................................ .. 10 G. Potential of DAS

- 444Ø6

- 6 to impact the weediness of other plants with which it can interbreed

................................ ................................ ................................ ................................ ................ 11 H. Potential changes to agricultural or cultivation practices

................................ ....................... 12 I. Potential impacts from transfer of genetic information to organisms with which DAS

- 444Ø6

- 6 cannot interbreed

................................ ................................ ................................ .................... 12 J. Conclusion

................................ ................................ ................................ ................................ 13 K. References

................................ ................................ ................................ ............................... 13Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean



### A. **Introduction**
Dow AgroSciences LLC ( DAS ) and M.S. Technologies LLC (MS Tech) ha ve petition ed APHIS (APHIS number 11

- 234

- 01p ) for a determination that genetically engineered (GE) soybean ( Glycine max ) event DAS

- 444Ø6

- 6 is unlikely to pose a plant pest risk (DAS and MS Tech 2011) and, therefore, should no longer be a regulated article under APHIS’ 7 Code of Federal Regulations ( CFR) part 340. APHIS administers 7 CFR part 340 under the authority of the plant pest provisions of the Plant Protection Act of 2000 1 . This plant pest risk assessment was conducted to determine whether DAS

- 444Ø6

- 6 is unlikely to pose a plant pest risk.

Event DAS

- 444Ø6

- 6 was produced by transformation of cotyledonary node explants of soybean ( Glycine max cv Maverick) with Agrobacterium tumefaciens . Because A. tumefaciens is a plant pest and some of the regulatory sequences (promoter from cassava vein mos aic virus and terminator from A. tumefaciens ) used to facilitate expression of the genes in soybean were derived from plant pests, this soybean has been considered a regulated article under APHIS regulations at 7 CFR part 340. Potential impacts considered in this Plant Pest Risk Assessment are those that pertain to the use of DAS

- 444Ø6

- 6 and its progeny in the absence of confinement. APHIS regulation 7 CFR 340.6(c) specifies the information needed for consideration in a petition for nonregulated status. APHIS will evaluate information submitted by the applicant , in addition to current literature, related to plant pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new enzymes, or changes to plant metabol ism, weediness of the regulated article, any impacts on the weediness of any other plant with which it can interbreed, potential changes to agricultural or cultivation practices, potential effects to non

target organisms, and transfer of genetic informatio n to organisms with which it cannot interbreed , to determine if DAS

- 444Ø6

- 6 is unlikely to pose a plant pest risk. If APHIS determines that a GE organism is not a plant pest risk, then APHIS has no regulatory authority over that organism.

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 o r plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant

. (D) A bacterium. (E) A fungus. (F) A viru s 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.” Page 2 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean



### B. **D evelopmen**  **t of DAS** 
**- 444Ø6** 

**- 6**  **herbicide t olerant**  2 

### s **oybean**
In the U.S. soybean was grown on 75.0 million acres in 2011 (Figure 1, USDA NASS 2011 a ) with a value of $29.6 billion in 2008/2009 (USDA ERS 2011 a ). Growers select soybean lines adapted to the different environmental and climatic features, operator’s education, weed and disease pressures, cost of seed and other inputs, technology fees, human safety, ease and flexibility of the productions system an d marketing reasons (USDA ERS 2002 ; Brookes 2011 ).

Figure 1 . Soybean production areas in the U.S. (USDA NASS 2011 b ). The presence of weeds in soybean fields can cause greater production losses than either insects or diseases (Gibson 2005; Oerke 2006). Before the development of effective herbicides for the selective control of weeds in soybeans in the early 1960’s, cultu ral practices including tillage, use of weed free seed, row spacing and crop rotation were the only ways to control weeds (Wax 1973). By 1987, over 30 herbicides were being used on soybeans (Jordan 1987). With the 1996 commercial introduction and rapid adoption of glyphosate tolerant soybeans, a major change in herbicide usage occurred with an increasing use of glyphosate concurrent with the increased planting of glyphosate tolerant soybeans and a decrease in use of other soybean herbicides (Figure 2 ; NR C 2010 ; Young 2006).

2 The applicant has described DAS

- 444Ø6

- 6 soybean as “herbicide tolerant” and historically APHI S has also referred to GE plants with diminished herbicide sensitivity as “herbicide tolerant.” However, the phenotype would fall under the Weed Science Society of America’s (WSSA) definition of “herbicide resistance” since DAS

- 444Ø6

- 6 has an inherited abi lity to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type variety (WSSA 1998). By the WSSA definition, “resi stance [to an herbicide] may be naturally occurring or induced by such techniques as genetic engineer ing or selection of variants produced by tissue culture or mutagenesis.” Herbicide tolerance, by the WSSA definition, only applies to plant species with an “inherent ability” to survive and reproduce after herbicide treatment. Page 3 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
Consequently, the diversity of herbicides used for weed management has declined in soybean (Table 1 ; Young 2006) resulting in weed species shifts (Johnson 2009). Deregulation of DAS

- 444Ø6

- 6 would provide soybean growers with addition al options for the post

- emergent control of both broadleaf and grass weeds. The integration of other herbicides with different modes of action with glyphosate has been encouraged to improve the duration of weed control, to enhance control of glyphosate to lerant weeds, to reduce the risk of developing glyphosate resistant weeds and to control glyphosate

- resistant weeds (WSSA 2010). This soybean product would also provide a potential remedy to the increased incidence of weed species that are more tolerant t o glyphosate (DAS and MS Tech 2011 ; NRC 2010 ; WSSA 2010).

Figure 2. Percent acreage of genetically engineered crops in the U.S. (USDA ERS 2011 b ).

Herbicide 1990 1995 2001 2006 2,4 D 3 10 4 2,4

- D Dimethlyl salt 3 Acetic acid (2,4 D) 7 2,4

- DB 1 Acifluorfen 3 Alachlor 13 4 Bentazon 16 12 1 Chloramben 1 Chlorimuron, ethyl 20 16 5 4 Clethodim 5 4 3 Clomazone 7 4 Cloransulam, methyl 5 1 Dimethenamid 1 Ethalfluralin 5 1 Page 4 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
Fenoxaprop 6 3 Fluazifop, P, butyl 6 10 3 1 Flumetsulam 2 Flumiclorac, pentyl 1 Fomesafen 2 4 7 2 Glyphosate 5 21 73 92 Imazamox 5 Imazaquin 16 15 2 1 Imazethapyr 11 44 9 3 Lactofen 1 5 1 Linuron 6 2 Metolachlor 10 7 Metribuzin 19 11 2 2 Paraquat 2 2 1 Pendimethalin 14 26 10 3 Quizalofop 3 6 S

- Metolachlor 1 Sethoxydim 4 7 1 Sulfentrazone 5 1 Sulfosate 3 1 Thifensulfuron 4 12 2 1 Tribenuron, methyl 1 Trifluralin 37 20 7 2 Table 1. Percent of U.S. Soybean Acres Treated with Herbicides in 1990, 1995, 2001 and 2006 ( USDA NASS 2010c)

DAS

- 444Ø6

- 6 is a GE soybean li ne that has been developed to increase tolerance to the herbicides 2,4

- D , glufosinate and glyphosate

. The introduced geneti c material results in the production of aryloxyalkanoat e dioxygenase

- 12 (AAD

- 12) that degrades the herbicide 2,4

- D into herbicida lly

- inactive 2,4

- dichlorophenol ( Müller 1999; Weste ndorf 2002 and 2003; Wright 2010a ) , phosphinothricin acetyl transferase (PAT) that metabolizes the L

- isomer 3 of glufosinate into non
- phytotoxic N

- acetyl

- L

- glufosinate (2

- acetamido

- 4

- methylphosphinico

butanoic acid) ( OECD 2002) , and modified 5

- enolpyruvylsh ikimate

- 3

- phosphate synthase ( EPSPS) that is insensitive to glyphosate ( Lebrun 1996; Lebrun 2003). If given non

regulated status , DAS

- 444Ø6

- 6 soybean would be the first soybean variety with increased tolerance to 2,4

- D , glufosinate , and glyphosate

.

3 The herbicide glufosinate, which is utilized as an ammonium salt in commercial formulation, consists of a racemic mixture of D and L enantiomers (Liu 2009). D

- glufosinate in the racemic D , L mixture is not acetylated by PAT and is not phytotoxic to plants ( Beriault 1999). Page 5 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

2,4

- D (2,4

- dichlorophenoxyacetic acid) is an herbicide in the phenoxy or phen oxyacetic acid family used for selective control of broa dleaf weeds since the mid

- 1940s in over 600 products in agricultural and residential applications ( US EPA 2005). The mode of action of 2,4

- D for broadleaf plants is unclear, but it is believed to function as a plant growth regulator 4 with synthetic auxin ho rmone
- like properties. When applied as an herbicide 2,4

- D causes abnormal cell division and growth le ading to plant injury and death.

Glufosinate (phosphinothricin; DL

- homoalanin

- 4

- yl(methyl) phosphinic acid) is a no n
selective foliar herbicide used for pre
- plant and post

- em ergent control of broadleaf plants and annual and perennial grasses ( OECD 2002; US EPA 2008)

. Glufosinate acts by inhibiting the enzyme glutamine synthetase, which leads to poisoning in plants because of the overproduction of ammonia

. Glufosinate was first registered by EPA for use in 2000 as a non
- selective foliar herbicide that is used for pre

- plant and post

- emergent control of broadleaf weeds ( US EPA 2008)

. EPA registration authorizes use on many crops including; apples, berries, canola, corn, cotton, currants, grapes, grass grown for seed, potatoes, rice, soybeans, sugar beets, and tree nuts and in non
- crop areas including lawns and residential areas ( US EPA 2008 ). The first soybean line containing a gluf osinate resistance trait produced through the use of biotechnology was granted nonregulated status in 1996 (USDA APHIS 2011). Glyphosate ( N

- (phosphonomethyl) glycine ) is a phosphonomethyl derivative of the amino acid glycine that inhibits the enzyme 5

- enolpyruvylshikama te

- 3

- phosphate synthase (EPSPS) resulting in the disruption of the pathway to synthesize aromatic amino acids ( Amrhein 1980; Steinrucken 1980 ). Glyphosate is widely used to control weeds in agricultural crops and
non
- agricultural site s and is registered for use on a variety of fruit, vegetable, and field crops as well as for aquatic and terrestrial uses (USEPA 2009)

. Glyphosate is also registered for use on glyphosate

- resis tant (transgenic) crop varieties such as canola, co rn , cotton, soybeans, alfalfa, and sugar beets. Labeled uses of glyphosate include over 100 terrestrial food crops as well as other non

- food sites including forestry, greenhouse, non

- crop, and residential

. The first soybean line containing a glyphosate resistance trait produced through the use of biotechnology was granted nonregulated status in 1994 (USDA APHIS 2011).



### C. **Descript ion of the m**  **odification**
Soybean DAS

- 444Ø6

- 6 was produced by transformatio n using disarmed Agrobacterium tumefaciens ( DAS and MS Tech 2011 , p. 21 ). Soybean (cultivar Maverick) cotyledonary nodes were infected with Agrobacterium strain EHA101 (Hood 198 6) containing plasmid

4 Plant Gr owth Regulators are synthetic plant hormones that regulate cellular processes, plant growth and development. Auxin compounds represent a class of hormones that along with other plant hormones determine patterns of plant development. Page 6 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
pDAB8264

. Plants containing the introduced DNA were selected based on growt h in the present of glufosinate

. The plasmid pDAB8264 contained three gene expression cassettes flanked by T

- DNA border sequences from the Agrobacterium Ti

- plasmid ( DAS and MS Tech 2011 , pp. 22 and 26

- 28 ).

Feature Name Length Description T

- DNA Border B 24 Required for transfer of T

- DNA insert from Agrobacterium tumefaciens into plant cells (Barker 1983) Intervening sequence 136 Non

- specific DNA sequences necessary for cloning

RB7 MAR 1166 Matrix attachment region from the Nicotiana tabacum rb

- 7

- 5A gene (Hall 1991) Intervening sequence 39 Non

- specific DNA sequences necessary for cloning

Histone H4A748 3′ UTR 661 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the histone H4A748 gene from Arabidopsis thaliana (Chaboute 1987) Intervening sequence 23 Non

- specific DNA sequences necessary for cloning
2mepsps 1338 Native 5

- enolpyruvylshikimate

- 3

- phosphate synthase gene from Zea mays with two mutations providing glyphosate tolerance (Lebrun 1996, Lebrun 2003) TPotp C 372 Optimized chloroplast transit peptide derived from maiz e and sunflower RuBisCO (Lebrun 1996, Lebrun 2003) Intervening sequence 4 Non

- specific DNA sequences necessary for cloning

Histone H4A748 promoter 1430 Promoter along with the 5′ untranslated region of the Histone H4A748 gene from Arabidopsis thaliana including an intron from the Histone 3 gene from Arabidopsis thaliana (Chaboute 1987) Intervening sequence 92 Non

- specific DNA sequences necessary for cloning
AtUbi10 promoter 1322 Promoter along with the 5' untranslated region and intron from the Arabidopsis thaliana polyubiquitin 10 (UBQ10) gene (Norris 1993) Intervening sequence 8 Non

- specific DNA sequences necessary for cloning

aad

- 12 882 Plant

- optimized version of an aryloxyalkanoate dioxygenase gene from Delftia acidovorans encoding an enzyme with an alpha ketoglutarate

- dependent dioxygenase activity which results in metabolic inactivation of the herbicide(s) (Wright 2009; Wright 2010) Intervening sequence 102 Non

- specific DNA sequences necessary for cloning

AtuORF23 3′ UTR 457 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 23 (ORF23) of plasmid pTi15955 from Agrobacterium tumefaciens (Barker 1983) Intervening sequence 114 Non

- specific DNA sequences necessary for cloning

Page 7 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

CsVMV promoter 517 Promoter along with the 5′ untranslated region derived from the Cassava Vein Mosaic virus (Verdaguer 1996) Intervening sequence 7 Non

- specific DNA sequences necessary for cloning

pat 552 Plant

- optimized version of phosphinothricin acetyltransferase (PAT) gene, isolated from Streptomyces viridochromogenes , encoding a protein that confers tolerance to glufosinate (Wohlleben 1988) Intervening sequence 102 Non

- specific DNA sequences necessary for cloning

AtuORF1 3′ UTR 704 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 1 (ORF1) of plasmid pTi15955 from Agrobacterium tumefaciens (Barker 1983) Intervening sequence 228 Sequence from Ti plasmid C58 (Zambryski 1982; Woo d 2001)

T

- DNA Border A 24 Required for transfer of T

- DNA insert from Agrobacterium tumefaciens into plant cells (Barker et al. , 1983) Intervening sequence 19 Sequence from Ti plasmid C58 (Zambryski 1982, Wood 2001)

T

- DNA Border A 24 Required for transfer of T

- DNA insert from Agrobacterium tumefaciens into plant cells, aiming to prevent vector DNA being transferred into plant genome (Barker 1983) Intervening sequence 287 Sequence from Ti plasmid pTi15955 (Barker 1983)

T

- DNA Border A 24 Required for transfer of T

- DNA insert from Agrobacterium tumefaciens into plant cells, aiming to prevent vector DNA being transferred into plant genome (Barker 1983)

Data from Southern blot analysis (DAS and MS Tech 2011, Table 3, Figures 6 & 7) demonstrate that DAS

- 444Ø6

- 6 contains a single copy of : (1) RB7 (Figures 8A & 8B); (2) histone H4748 promoter (Figures 10A, 10B & 18A), 2 mepsps gene (Figures 15A, 15B, 15C & 18B) and histone H4A748 terminator (Figures 9A, 9B & 18C); (3 ), polyubiquitin promoter (Figure s 11A, 11B & 19A), aad

- 12 gene (Figures 16 A, 16B, 16C & 19B) and ORF 23 terminator (Figure s 12A, 12B & 19C); (4 ) CsVMV promoter (Figures 13A, 13B & 20A), pat gene (Figures 17A, 17B, 17C & 20B) and ORF 1 terminator (Figures 14A, 14B & 20C). No plasmid backbone sequences were detected (Figure s 21A, 21B, 22A, 22B, 23A, 23B, 24A, 24B, 25A and 25B).



### D
**. Pot**  **ential for DAS**

**- 444Ø6** 

**- 6 to have altered disease and pest** 
**s usceptibilities** 

Page 8 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

APHIS assessed whether DAS

- 444Ø6

- 6 is likely to have significantly altered di sease and pest susceptibility. This assessment encompassed a consideration of the introduced trait and disease and pest susceptibility data from DAS

- 444Ø6

- 6 field trials.

DAS

- 444Ø6

- 6 w as released in the U.S. from 2009 through 2011 in twenty five locations across a diverse range of environmental conditions representative of where DAS

- 444Ø6

- 6 is expected to be grown (DAS and MS Tech 2011, pp. 88 & 194). DAS and MS Tech used well

- established qualitative and quantitative techniques to observe insect and disease damage (DAS and MS Tech 2011, pp. 78

- 89)

. The following d isease and insect pests were evaluated in the field: brown spot, Carlavirus, frogeye leaf spot, rust, sudden death syndrome; aphids, bean leaf beetles, green cloverworm, grasshoppers, g round beetles, ladybugs, leaf hoppers, Lepidoptera spp., and stink bugs. In 2010 the c ombined

- site analysis across ten locations found no statistically significant differences for seed germination and emergence, seedling vigor, days to flower ing , disease incidence, insect damage, days to maturity, lodging, plant height, final population, number of pods, number seeds per plant , shattering, yield and seed weight (DAS and MS Tech 2011, pp. 83

- 84 )

. Data were collected on pest and disease damage across twenty five release sites in 2009 and 2010 (DAS and MS Tech 2011, p. 78)

. In all cases , no significant differences between DAS

- 444Ø6

- 6 and the conventional parental line Maverick were observed. No qualitative or quantitative observations indicated any bi ologically meaningful differences from control lines or differences outside the range of different soybean varieties

. The description of the introduced genetic elements, expression of the gene products and their functions of DAS

- 444Ø6

- 6 have been summari zed above. The A. tumefaciens transformed plants used in the generation of DAS

- 444Ø6

- 6 were treated with an antibiotic to kill the A. tumefaciens cells. DNA sequences derived from plant pests that were incorporated in DAS

444Ø6

- 6 do not result in the prod uction of infectious agents or disease symptoms in plants, and so it is unlikely that DAS

- 444Ø6

- 6 could pose a plant pest risk. The description of the introduced genetic elements, expression of the gene products and their functions in DAS

444Ø6

- 6 has been summarized above. Given the interactions between the environment, the genetic backgrounds of the cultivars used and some inherent genetic variability within soybean varieties, APHIS concludes that these results do not indicate an increased pest risk. E xpression of AAD

- 12 , EPSPS and PAT in event DAS

- 444Ø6

- 6 soybean is not expected to cause plant disease or influence susceptibility of DAS

- 444Ø6

- 6 or its progeny to diseases or other pests.

Page 9 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean



### E. Potential for effect on non

**- target organisms, including those**  **b eneficial to agriculture** 
There is no reason to believe that deleterious effects or significant impacts on non

- target organisms, including beneficial organisms, would result from the cultivation of DAS

- 444Ø6

- 6. Field observations of DAS

- 444Ø6

- 6 (DAS and MS Tech 2011, section 7) re vealed no negative effects on non

- target organisms, suggesting that the production of the ADD

- 12, PAT and EPSPS proteins in the plant tissues are not toxic to organisms. The introduced genetic material does not result in the production of novel proteins, enzymes, or metabolites in the plant that are known to have toxic properties. The lack of known toxicity of ADD

- 12, PAT and EPSPS suggests no potential for deleterious effects on beneficial organisms such as bees and earthworms. The use of glyphosate, 2,4

- D and glufosinate herbicides in the cultivation of DAS

- 444Ø6

- 6 or its offspring is regulated by EPA under its existing regulations for the registration of pesticide use. EPA considers the impacts on the environment , including effects on non

- target organi sms in establishing residue tolerances for glyphosate, 2,4

- D and glufosinate tolerant lines (USEPA 1993 ; USEPA 2005 ; USEPA 1997). APHIS has not identified any other potential mechanisms for d eleterious effects on
non
- target organisms

.


### F. **Potential for enha**  **nced weediness or invasiveness**

APHIS assessed whether DAS

- 444Ø6

- 6 soybean is any more likely to become a weed than the non

- transgenic recipient soybean line or other soybean lines currently cultivated. The assessment encompasses a consideration of the ba sic biology of soybean and an evaluation of unique characteristics of DAS

- 444Ø6

- 6 soybean. Weediness for the purposes of this part of the plant pest risk assessment is an attribute, which causes a crop to act as a weed due to the addition of genes, in com parison to the non

- GE comparator (parental line Maverick). If the fitness of DAS

- 444Ø6

- 6 soybean improves in natural or agricultural ecosystems due to the inserted DNA, the potential for weediness could increase. The following analysis of the inserted DN A is intended to document that DAS

444Ø6

- 6 soybean has a negligible likelihood of increased weediness. In the U.S., soybean is not listed as a weed in the major weed references (Crockett 1977; Holm 1979; Muenscher 1980 ) nor is it designated as n oxious weed by the feder al government (USDA NRCS 2012 a ). Soybean 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 specie s in new or diverse landscapes, or the ability to compete w ell with native vegetation. Furthermore, m ature soybean seeds
Page 10 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
have no innate dormancy, are sensitive to cold, are not expected to survive in freezing winter conditions and do not reproduce vegetati vely ( Hermann 1962; OECD 2000; Pad gette 1996; Raper Jr. & Kramer 1987).

From 2009 through 2011 , DAS and MS Tech conducted field trials to evaluate phenotypic characteristics comparing DAS

- 444Ø6

- 6 with the non

- transge nic soybean parental variety Maverick (DAS a nd MS Tech 2011, pp. 88 & 194). Results on growth characteristics, seed production and germination indicate that DAS

- 444Ø6

- 6 is not significantly different from its comparators (DAS and MS Tech 2011, pp. 79

- 85). No biologically meaningful differe nces were observed across sites between DAS

- 444Ø6

- 6 and the parental variety Maverick

. To increase weediness of the soybean plant there would have to be selection pressure on the line (Tiedge 1989). Because neither 2,4

- D, glufosinate or glyphosate will affect the survival of DAS

- 444Ø6

- 6 and because soybean is not itself weedy, this type of selection pressure does not now and is unlikely to exist.

There is no indication that DAS

- 444Ø6

- 6 possesses a selective advantage that would result in increased weediness. DAS

- 444Ø6

- 6 lacks the ability to persist as a troublesome weed, and there would be no significant impact on current weed management practices for soybean cultivation.



### G. Potential of DAS

**- 444Ø6** 

**- 6 to impact the weediness of other plants**  **with whic h it can i**  **nterbreed**
The genus Glycine , a member of the Fabace ae (= Leguminosae or pea family), consists of two subgenera, soja and glycine (OECD 2000 ; USDA NRCS 2012 b )

. Perennial species in the subgenus glycine d o not occur in the U.S. (USDA NRCS 2012 b ) , except in the U.S. territories in the South Pacific (Hymowitz & Singh 1987). 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 (L.) Merr. , t he cultivated soybean. G. soja and G.
max do not occur naturally in the U.S. ( Hermann 1962; Hymowitz 1987; USDA NRCS 2012 b)

. Hybrids from crosses between the subspecies have generally been sterile, and further progeny have only been obtained with extreme difficulty (OECD 2000).

Cultivated s oybean is highly self

- pollinat ing (Ahrent 1994). When soybean plants are grown directly adjacent to other soybean plans, t he amount of natural c ross pollination has gene rally been found to be 0.5

- 1 percent (Fehr 19 80 ; OECD 2000 ) although high er values (2.5 percent) occur in some varieties (Abud 2007 )

. Out crossing ca n be reduced to 0

– 0.01 percent with a s eparation distance of 10 meters (Abud 2007).

Page 11 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
The cultivated soybean, G. max , lacks sexually compatible wild relatives in the U.S. and its territories. Consequently, there is no potential for gene flow from cultivated soybean plants to wild relatives in the U.S. Therefore, it is not likely that gene flow and introgression will
oc cur between DAS

- 444Ø6

- 6 and other species of soybea n. APHIS has determined that any adverse consequences of gene flow from DAS

- 444Ø6

- 6 soybean to wild or weedy species in the United States are highly unlikely.


### H. Potential changes to agricultural or culti **vation practices**
None of the management practices currently employed for soybean production is expected to change if DAS

- 444Ø6

- 6 is determined to be no longer subject to the regulatory requirements of 7 CFR part 340 or the plant pest provisions of the Plant Protection Act. DAS and MS Tech’s (2011) studies demonstrate that the agronomic characteristics and cultivation practices employed when growing DAS

- 444Ø6

- 6 soybean are essentially indistinguishable from practices used to grow other soybean varieties, including other he r bicide

- tolerant varieties (DAS and MS Tech 2011 , Section 7.4)

. Although DAS

- 444Ø6

- 6 soybean might be expected to replace other varieties of soybean currently cultivated, additional acreage is not expected to be developed to accommodate the cultivation of DAS

- 444Ø6

- 6 (DAS and MS Tech 2011). DAS

- 444Ø6

- 6 is comparable to currently available soybean varieties in terms of resistance to insects and disease (DAS and MS Tech 2011 , pp. 78

- 89). Therefore, no changes are expected for insect and disease control prac tices with DAS

- 444Ø6

- 6. Because agricultural and cultivation practices would not be significantly different than that of conventional soybean , APHIS does not foresee changes in on insects or diseases damage or control measures employed due to agricultural or cultivation practices with DAS

- 444Ø6

- 6.


### I. Potential impacts from transfer of genetic information to organisms **with which DAS**

**- 444Ø6** 

**- 6 cannot interbreed** 
APHIS examined the potential for the new genetic m aterial inserted into DAS

- 444Ø6

- 6 to be horizont ally transferred to other organisms without sexual reproduction and whether such an event could lead directly or indirectly to disease, damage, injury or harm to plants. Horizontal gene transfer and expression of DNA from a plant species to other species i s highly unlikely to occur based on the following reasons
. The horizontal gene transfer (HGT) between unrelated organisms is one of the most intensively studied fields of science

. Horizontal gene transfer and expression of DNA from a plant species to bac teria or animal species is unlikely to occur (Keese 2008)

.

Page 12 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

1. M any genomes (or parts thereof) from bacteria that are closely associated with plants have been sequenced , including Agrobacterium and Rhizobium (K aneko 20 00; Kaneko 2002 ; Wood 2001 ). There is no evidence that these organisms contain genes derived from plants. Therefore the likelihood of a ny impact or new horizontal gene transfer that is not already capable of taking place in the soil is extremely unlikely. 2. No evidence has been ident ified for any mechanism by whi c h soybean genes could be transferred to humans or animals, or any evidence that such gene transfer has occurred for any plant species during evolutionary history, despite animals and humans eating large quantities of plant DNA. I n cases where review of sequence data impl ied that horizontal gene transfer occurred, these events are inferred to occur on an evolutionary time scale on the order of m illions of years (Brown 2003 ; Koonin 2001 ). 3. 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. 4. FDA has evaluated horizontal gene transfer from the use of antibiotic resistance marker genes, and co ncluded 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 extremely unlikely (FDA 1998). Therefore APHIS concludes that horizont al gene transfer is highly unlikely to occur and thus poses no significant plant pest risk.


### J. Conclusion
APHIS has prepared this plant pest risk assessment in order to determine if event DAS

444Ø6

- 6 is likely to pose a plant pest risk. Based on the information provided by the applicant and the lack of atypical responses to disease or plant pests in the field, weedy characteristics of the DAS

- 444Ø6

- 6 or other plants with which it can interbreed, changes to agricultural or cultivation prac tices, effects on non

- targets or beneficial organisms in the agro
- ecosystem, indirect effects on other agricultural products and the unlikelihood of horizontal gene transfer, APHIS has concluded that soybean event DAS

- 444Ø6

- 6 is highly unlikely to pose a p lant pest risk.



### K. References
Abranches, R., Shultz, R.W., Thompson, W.F., Allen, G.C. (2005) Matrix attachment regions and regulated transcription increase and stabilize transgene expression. Plant Biotechnology Journal 3:535

- 543.
Abud, S., de Souza, P .I.M., Vianna, G.R., Leonardecz, E., Moreira, C.T., Faleiro, F.G., Júnior, J.N., Monteiro, P.M.F.O., Rech, E.L., Aragão F.J.L. (2007) Gene flow from Page 13 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
transgenic to nontransgenic soybean plants in the Cerrado region of Brazil. Genetics and Molecular Research 6:445

- 452.
Ahrent, D.K., Caviness, C.E. (1994) Natural cross

- pollination of twelve cultivars in Arkansas. Crop Science 34:376

- 378. Allen, G.C., Spiker, S., Thompson, W.F. (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Molecular Biology 43:361

- 376.
Amrhein, N., Deus, B., Gehrke, P., Steinrucken, H.C. (1980) The site of the inhibition of the shikimate pathway by glyphosate. II. Interference of glyphosate with chorismate formation in vivo and in vitro . Plant Physiol ogy 66:830

- 834.
Baker, H. B. (1965) “ Characteristics and modes of origin of weeds” p p. 147

- 169 In H. G. Baker and G. L. Stebbins (eds.) The Genetics of Colonizing Species. Academic Press, London.

Barker , R.F. , Idler, K.B. , Thompson, D.V. , Kemp, J.D. (1983) Nucleotide sequence of the TDNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol ecular Biology 2:335

- 350. Beriault, J.N., Horsman, G.P., Devine, M. (1999) Phloem transport of D,L

- glufosinate and acetyl

- L

- glufosinat e in glufosinate

- resistant and

- susceptible Brassica napus. Plant Physiology 121:619

- 627. Brookes, G., Barfoot, P. (2011) The income and production effects of biotech crops globally 1996

- 2009. International Journal of Biotechnology 12:1

- 49.

Brown , J

. R . (2003) Ancient horizontal gene transfer. Nature Reviews Genetics 4:121

- 132.
Chaboute, M.

- E., Chaubet, N., Phili pps, G., Ehling, M., Gigot, C. (1987) Genomic organization and nucleotide sequences of two histone H3 and two histone H4 gene s of Arabidopsis thaliana . Plant Molecular Biology 8: 179

- 191. Crockett, L. (1977) Wildly Successful Plants: North American Weeds. University of Hawaii Press, Honolulu, Hawaii. 609 pp. Dow AgroSciences LLC and M.S. Technologies LLC (2011) Petition for Determination of Nonregulated Status for Herbicide Tolerant DAS

- 444Ø6

- 6 Soybean. Submitted by M.S. Krieger. Indianapolis, IN

. http://www.aphis.usda.gov/biotechnology/not_reg. html

Page 14 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
FDA (1998) Guidance for Industry: Use of Antibiotic Resistance Marker Genes in Transgenic Plants. pp. 28.
Fehr, W .R., Hadley, H.H. (1980) Hybridization of Crop Plants

. American Society of Agronomy and Crop Science Society of America, Madison, Wisco nsin. (p. 592). Gianessi, L., Reigner, N. (2006) Pesticide Use in U.S. Crop Production: 2002. CropLife Foundation. Retrieved December , 2011, from http://www.croplifefoundation.org/

. Gibson, K.D., Johnso n, W.G., Hillger, D.E. (2005) Farmer perceptions of problematic corn and soybean weeds in Indiana. Weed Technology 19:1065

- 1070. Hall Jr., G., Allen, G.C., Loer, D.S., Thompson, W.F. (1991) Nuclear scaffolds and scaffold

attachment regions in higher plants. Proceedings National Academy of Sciences 88:9320

- 9324. Hermann, F.J. (1962) A revision of the genus Glycine and its immediate allies. Technical Bulletin No. 1268. United State Department of Agriculture, Agricultural Research Service.
Holm, L., Pancho, J. V., Herbarger, J. P., Plucknett, D. L. (1979) A Geographical Atlas of World Weeds. John Wiley and Sons, New York. 391 pp. Hood, E.E., Helmer, G.L., Fraley, R.T., Chilton, M.

- D. (1986 ) The h ypervirulence of Agrobacterium tumefaciens A281 is e ncoded in a region of pTiBo542 outside of T

- DNA. Journal of Bacteriology 168 :1291

- 1301. Hymowitz, T., Singh, R.J. (1987) “Taxonomy and Sp eciation” In Soybeans: Improvement, Production, and Uses

- Second Edition. American Society of Agronomy.
Johnson, W.G., Davis, V.M., Kruger, G.R., Weller, S.C. (2009) Influence of glyphosate

resistant cropping systems on weed glyphosate

- resist ant weed populations. European Journal of Agronomy 31:162

- 172.

Jordan , T.N., Coble, H.D., Wax, L.M. (1987) “Weed Control” In S oybeans: Improvement, Production, and Uses

- Second Edition. American Society of Agronomy.

Kaneko T

. et al. (2000) Complete genome structure of the nitrogen

- fixing symbiotic bacterium Mesorhizobium loti . DNA Research 7:331

- 338.

Page 15 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

Kaneko T

. et al. (2002) Co mplete genomic sequence of nitrogen

- fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Research 9:189

- 197

.
Keese , P

. (2008) Risks from GMOs due to horizontal gene transfer. Environmental Biosafety Research 7:123

- 149.
Koonin, E.V., Makarova, K.S., Aravind, L. (2001) Horizontal gene transfer in prokaryotes: quantification and classification . Annual Review of Microbiology 55:709

- 742. Lebrun, M., Leroux, B., Sailland, A. (1996) Chimeric gene for the transformation of plants. U.S. Patent No. 5, 510,471.
Lebrun, M., Sailland, A ., Freyssinet, M., Degryse, E. (2003) Mutated 5

- enolpyruvylshikimate

- 3

- phosphate synthase, gene coding for said protein and transformed plants containing said gene. U.S. Patent No. 6,566,587.

Liu , W., Ye, J., Jin, M. (2009) Enantioselective phytoeffects of chiral pesticides. J. Agric. Food Chem. 57:2087

- 2095.
Muenscher, W. C. (1955 ) Weeds. Second Edition. Cornell University Press, New York and London . (p. 545).
Müller, R.H.; Jorks, S.; Kleinsteuber , S.; Babel, W. (1999) Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4

- D and MCPA. Microbiological Research 154:241

- 246.
Norris, S.R. , Meyer, S.E. , Callis, J. (199 3) The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expressio n. Plant Molecular Biology 21 :895

- 906. NRC (National Research Council) (2010) Environmental Impacts of Genetical ly Engineered Crops at the Farm Level. p p. 2

- 1 to 2

- 45. In Impact of Genetically Engineered Crops on Farm Sustainability in the United States. The National Academies Press. Washington, D.C.

OECD Organization for Economic Co

- oper ation and Development Conse nsus (2000) Document on the Biology of Glycine max (L.) Merr. (Soybean).

OECD (2002) Mod ule II: Phosphinothricin Series on Harmonization of Regulatory Oversight in Biotechnology, No. 25.
Page 16 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

Oerke, E. C. (2006) Crop losses to pests. Journal of Agricultural Science 144:31

- 43. Padgette, S.R., Re, D.B., Barry, G.F., Eichholtz, D.E., Delannay, X., Fuchs, R.L., K ishore, G.M., Fraley, R.T. (199 6 ) “ New weed control opportunities: development of soybe ans with a Roundup Ready gene ” p p
. 53

- 84. In S.O. Duke (ed.). Herbicide Resistant Crops

Agricultural, Environmental, Economic, Regulatory, and Technical Aspects. CRC Press, Boca Raton, Florida. Raper, Jr., D.C., Kramer, P.J. (1987) “Stress Physiology” pp. 589

- 641. In J.R. Wilcox (ed.). Soybean: Im provement, Production, and Uses. American Society of Agronomy. Spiker, S., Thompson, W.F. (1996) Nuclear matrix attachment regions and transgene expression in plants. Plant Physiology 110:15

- 21. Steinrucken, H.C., Amrhein, N. (1980) The herbicide glyphos ate is a potent inhibitor of 5

- enolpyruvylshikimic acid

- 3

- phosphate synthase. Biochemical and Biophysical Research Communications 94:1207:1212. Tiedje, J.M., Colwell, R.K., Grossman, Y.L., Hodson, R.E., Lenski, R.E., Mack, R.N., Regal, P.J. (1989) The pla nned introduction of genetically engineered organisms: ecological considerations and recommendations. Ecology 70:298

- 315. USDA APHIS (2011) United States Department of Agriculture, Animal and Plant Health Inspection Service. Petitions for Nonregulated Sta tus Granted or Pending by APHIS. Retrieved December 2011, from http://www.aphis.usda.gov/biotechnology/not_reg.html

USDA ERS (2002) United States Department of Agriculture, Economic Research Service. Adoption of Bioengineered Crops, Agricultural Economic Report No. 810. J. Fernandez

Cornejo and W.D. McBride. Retrieved December , 2011, from http://www.ers.usda.gov/publications/aer810/aer810.pdf
USDA ERS (2011 a ) United States Department of Agriculture, Economic Research Service. Soybeans and Oil Crops. Retrieved December , 20 11, from http://www.ers.usda.gov/Briefing/SoybeansOilCrops/

USDA ERS (2011 b ) United States Department of Agriculture, Economic Research Service. Adoption of Genetically Engineered Crops in the U.S.: Soybeans Varieties. Retrieved December , 2011, from http://www.ers.usda.gov/Data/BiotechCrops/
Page 17 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

USDA NASS (2011 a) United States Department of Agriculture, National Agricultural Statistics Service. National Statistics for Soybean. Soybean Acres Planted. Retrieved December, 2011 , from http://www.nass.usda.gov/Statistics_by_Subject/result.php?3E9BD064

- FBF6

- 30AD

8530

- 513C0E23C55C§or=CROPS&group=FIELD%20CROPS&comm=SOYBEANS
USDA NASS (2011 b) United States Departmen t of Agriculture, National Ag ricultural Statistics Service. National Statistics for Soybean. Soybean Acres Planted. Retrieved December, 2011 , from http://www.nass .usda.gov/Charts_and_Maps/Crops_County/pdf/SB

- PL10

- RGBChor.pdf
USDA NASS (2011 c ) United States Department of Agriculture, National Agricultural Statistics Service. Agricultural Chemical Use Database. Retrieved December , 2011, from http://www.pestmanagement.info/nass/
USDA NRCS (2012 a ) United States Department of Agriculture, Natural Resources Conservation Service. Invasive and noxious weeds. Retrieved May , 2012 , from http://plants.usda.gov/java/noxiousDriver

USDA NRCS (2012 b ) United States Department of Agricultu r e, Natural Resources Conservation Service. The PLANTS Database. National Plant Data Team. Retrieved January 2012, from http://plants.usda.gov/java/

USEPA (1993) United States Environmental Protection Agency. Reregistration Eligibility Decision (RED) Glyphosate. 738

- R

- 93

- 014. Retrieved December, 2011, from http://www.epa.gov/oppsrrd1/REDs/old_reds/glyphosate.pdf
USEPA (1997) United State s Environmental Protection Agency. Phosphinothricin acetylt ransferase and the genetic mater ial necessary for its production in all plants; exemption from the requirement of a tolerance on all raw agricultural commodities. 62 Federal Register 17717

- 17720. USEPA (2005) United States Environmental Protection Agency. Reregistration Eligibility Decision for 2,4

- D. Retrieved December, 2011, from http://www.epa.gov/oppsrrd1/REDs/24d_red.pdf

Page 18 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean

USEPA (2008 ) United States Environmental Protection Agency

. Glufosinate Summary Document. Retrieved December, 2011, from http://www.epa.gov/oppsrrd1/registration_review/glufosinate_ammonium/index.htm

USEPA (2009) United States Environm ental Protection Agency

. Glyphosate Summary Document Registration Review: Initial Docket. Retrieved December , 2011, from http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPP

- 2009

- 0361

- 0003;oldLink=false Verdaguer, B. , Kochko de, A. , Beachy, R. N., Fauquet, C. (1996) Isolation and expre ssion in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter. Plant Molecular Biology 31:1129

- 1139. Wax, L.M., (1973) “Weed Control” in Soybeans: Improvement, Production, and Uses. Amer ican Society of Agronomy. Caldwell, B.E. (ed.) pp. 417

- 457.

Wen , A., Fegan, M. , Hayward , C. , Chakrabory, S. (1999) Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov. Westendorf, A.; Benndorf, D.; Müller, R.; Babel, W. (2002) The two enantiospecific dichlorprop/ α - ketoglutarate

- dioxygenases from Delftia acidovorans MC1

- protein and sequence data of RdpA and SdpA

. Microbiological Research 157:317

- 322.
Westendorf, A.; Mül ler, R.H.; Babel, W. ( 2003) Purification and characterization of the enantiospecific dioxygenases from Delftia acidovorans MC1 initiating the degradation of phenoxypropionates and phenoxyacetate herbicides. Acta Biotechnologica 23: 3

- 17.
Wood, D.W. et al. (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294 :2317

- 2323.
Wohlleben, W. , Arnold, W., Broer, I., Hillemann, D., Strauch, E. and Pühler, A. (1988) Nucleotide sequences of the phosphinothricin N

- acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum . Gene 70: 25

- 37. Wright, T.R., Lira, J.M., Merlo, D.J., Arnold, N.L. (2010a) Novel herbicide resistance genes. U.S. Patent No. 7,838,733 B2

.

Page 19 of 20Plant Pest Risk Assessment for Herbicide
- Tolerant DAS
- 444Ø6

- 6 Soybean
Wright, T.R., Shan, G., Walsh, T.A., Lira, J.M., Cui, C., Song, P., Zhuang, M., Arnold, N.L., Lin, G., Yau, K., Russell, S.M., Cicchillo, R.M., Peterson, M.A., Simpson, D.M., Zhou, N., Ponsamuel, J., Zhang, Z. (2010b) Robust crop resistance to broadleaf an d grass herbicides provided by aryloxyalkanoate dioxygenase transgenes. Proceedings of the National Academy of Sciences 107:20240

- 20245. WSSA Weed Science Society of America (1998) “Herbicide Resistance” and “Herbicide Tolerance” defined. Weed Technology . 12:789

. WSSA Weed Science Society of America (2010) WSSA Supports NRC Findings on Wee d Control. Retrieved January , 2012, from http://www.wssa.net/WSSA/Information/WSSA%20position%20paper%20on%20herbic ide%20resistance%205

- 27

- 2010.pdf Young, B.G. (2006) Changes in herbicide use patterns and production pract ices resulting from glyphosate

- resistant crops. Weed Technology 20:301

- 307

.

Page 20 of 20