2 

## Table of Contents

Table of Contents .............................................................................................. 2 Plant Pest Risk Assessment of Bayer CropScience TwinLinkTM Cotton ......... 3

A. Introduction ..................................................................................... 3

B. History of Development of TwinLinkTM Cotton............................. 3

1. Description of the modifications ..................................................... 4

C. Plant Pest Risk Assessment ............................................................ 5

1. Potential impacts of altered dise ase and pest susc eptibilities ......... 6

2. Potential impacts from outcrossing of the BCS TwinLinkTM cotton

to wild relatives ............................................................................... 7

3. Potential impacts based on th e relative weediness of BCS

TwinLinkTM cotton. ......................................................................... 9

4. Potential impacts on target a nd nontarget organisms, including beneficial organisms. .................................................................... 10

5. Potential impacts from transferring genetic information from BCS TwinLinkTM cotton to organisms w ith which it cannot interbreed.

11

D. Conclusion .................................................................................... 12

E. References ..................................................................................... 12

## Plant Pest Risk Assessment of Bayer CropScience
TwinLinkTM Cotton

A. Introduction

Bayer CropScience (BCS) has petitioned Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture for a determination that TwinLinkTM cotton is unlikely to pose a plant pest risk and, therefor e, 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 pl ant pest provisions of the Plant Protection Act of 2000 1 . This plant pest risk assessment was conducted to determine if COT67B cotton is unl ikely to pose a plant pest risk.

Bayer CropScience (BCS) has developed cotton ( Gossypium hirsutum ) plants that express two insecticidal proteins, Cry1Ab and Cr y2Ae, from a common soil bacterium, Bacillus thuringiensis (Bt) and also expresses the bar gene that confers resistance to glufosinate ammonium herbicide

from Streptomyces hygroscopicus . The Cry1Ab and Cry2Ae protein are effective in controlling lepidopteran larvae such as bollworm (CBW, Helicoverpa zea ), tobacco budworm (TBW,

Heliothis virenscens ) larvae and fall armyworm (FAW, Spodoptera frugiperda ) which are common pests of cotton. In addition to the Cry1Ab and Cry2Ae proteins, TwinLinkTM cotton also contains the PAT (phosphinothricin-a cetyl-transferase) enzyme, encoded by the bar gene. This is the same protein that is expre ssed in Bayer CropScience Liberty Link cotton® (LLCotton25) that confers to the plant tole rance to the herbicide glufosinate ammonium.



### B. History of Development of TwinLinkTM Cotton

In 2002, the first phosphinothricin (glufosinate )-tolerant cotton, event GHB119; Aventis CropScience cotton designated Liberty Link cotto n® (LLcotton25) was determined by APHIS to be no longer subject to the regulatory require ments of 7 CFR Part 340 or the plant pest provisions of the Plant Protection Act. This ev ent involved cotton that was transformed with a binary plasmid vector carry the bar gene construct within a di sarmed transfer DNA (T-DNA)

from Agrobacterium tumefaciens. Aventis CropScience (now Ba yer CropScience) completed its food safety consultation with the U.S. Food and Drug Administration (FDA) on Liberty Link cotton® in 2003 (http://www.accessdata.fda.gov ; BNF 000086). Tolerance exemptions have been granted for the plant-incorporated protect ant in Liberty Link cotton® (event LLcotton25) and the genetic material necessary for its production. On April 11 1997, the Environmental Protection Agency (EPA) granted exemptions from the requirement of a to lerance for residues of the plant-pesticide phosphinothr icin-acetyl-transferase prot ein in or on food and feed commodities of cotton (62 FR 17719; 40 CFR 180.473). The Australia and New Zealand Food

1 Section 403 (14) of the Plant Protection Ac t (7USC Sec 7702(14)) defines plant pest as: “Plant Pest - The term “plant pest” means any living stage of any of the following that can directly or indirectly injure, cau se damage to, or cause disease in any plant or plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A f ungus. (F) A virus or viroid. (G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the articles speci fied in the preceding subparagraphs.”4 Authority (ANZFA) also completed a Toxicolo gical Review and Risk Assessment, and a Technical Report was published in 2005 (ANZFA Application A533, 2005; http://www.foodstandards.gov.au). ANZFA conc luded that Liberty Link cotton® was comparable to non-genetically engineered ( non-GE) cotton in terms of their safety and nutritional adequacy. In 2004, the Pl ant Biosafety Office (PBO) of the Canadian Food Inspection Agency (CFIA) concluded that Bayer CropSci ence’s Liberty Link cotton® was substantially equivalent to currently grown cotton, in term s of their potential e nvironmental impact and livestock feed safety and the nove l traits would not have any s ubstantial negative effect on the environment (CFIA Decision Document DD2004-49; http://www.inspection.gc.ca).

Bayer CropScience has now developed the tran sgenic cotton, TwinLinkTM, by crossing cotton event GHB119 that expresses both the Cry2Ae prot ein and phosphinothricin-acetyl-transferase (found in LiberyLink® cotton) proteins now with a newly developed co tton line (event 304-40) that expresses both Cry1Ab protein and phosphinothricin-acetyl-transferase (found in LiberyLink® cotton) proteins. TwinLinkTM cotton ha s been developed by BCS as an alternative

insect resistant and herbicid e tolerant cotton product.

TwinLinkTM cotton has been field tested under APHIS regulations since 2005. Data were provided in the petition for field trials complete d prior to the petition submission. Field test reports can be found in the BCS TwinLinkTM cotton petition in Appendix 1, p.102 (Bayer CropScience 2010).

On December 22 2008, BCS submitted a request that the Environmental Protection Agency (EPA) grant an exemption from the requirement of a tolerance for re sidues of the plantincorporated protectant (PIP), Bacillus thuringiensis Cry2Ae and Cry1Ab insect control proteins and the genetic material necessary for its pr oduction, in or on all food commodities. BCS has also submitted a food safety summary to FDA indicating that food and feed derived from

TwinLinkTM cotton are as safe and nutritious as food and feed derived fro m conventional cotton.



### 1. Description of the modifications

TwinLinkTM cotton contains the stably integrated genes cry 1Ab, cry 2Ae and bar , which encode respectively the Cry1Ab, Cry2Ae and PAT pr oteins. “The genes were introduced by Agrobacterium -mediated gene transfer. Southern blot analyses show TwinLink cotton contains one complete copy of the cry1Ab and cry2Ae genes, and 2 copies of the bar gene” (pg 2, Bayer CropScience 2010).

The two parent lines that were conventionally crossed to produce Twin LinkTM cotton contain the following genetic components (also descri bed in Bayer CropScience 2010 on page 21):

Cry2Ae + PAT (event GHB119) has been geneti cally engineered to contain the following transgene fragments:

Left border (LB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988).
3 ́nos: sequence including the 3’ untranslated re gion of the nopaline synthase gene from the T-DNA of pTiT37 in counter clockwise di rection (Depicker, Stachel et al. 1982)5 bar ; the coding sequence of the phosphinot hricin acetyltransferase gene of Streptomyces hygroscopicus in counter clockwise direction (De Block, Botterman et al. 1987)

Pcsvmv XYZ sequence including the promoter region of the Cassava Vein Mosaic Virus in counter clockwise direction (V erdaguer, De Kochko et al. 1996)

P35S2 sequence including the promoter region from Cauliflower Mosaic Virus 35S transcript in clockwise dir ection (Odell, Nagy et al. 1985)

5'cab22L sequence including the leader sequenc e of the chlorophyl a/b binding protein gene from Petunia hybrida in clockwise direction (Harpster, Townsend et al. 1988)

TPssuAt coding sequence of the transit pep tide of the ribulose-1,5-biphosphate carboxylase small subunit gene ats1A of Arabidopsis thaliana in clockwise direction (De Almeida, Gosselé et al. 1989) Cry2Ae the coding sequence of an ins ecticidal protein gene of Bacillus thuringiensis

, adapted to cotton codon usage in clockwise direction

3'35S sequence including the 3' untranslated regi on of the 35S transcript of Cauliflower

Mosaic Virus in clockwise direct ion (Sanfaçon, Brodman et al. 1991) Right border (RB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988)

Cry1Ab + PAT (event 304-40) has been geneti cally engineered to contain the following transgene fragments:

Right border (RB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988)

3’ me1 sequence that includes the 3’ untrans lated region of the NADP-malic enzyme gene of Flaveria bidentis (yellowtop) in counter cloc kwise direction (Marshall, Stubbs et al. 1996) Cry1Ab sequence from B. thuringiensis berliner 1715 in counter clockwise direction (Höfte, de Greve et al. 1986)

5’e1 sequence that includes the leader sequence of the tapetum specific E1 gene (GE1) of Oryza sativa (rice) in counter clockwise direction (Michiels F. 1992)

Ps7s7 sequence including the duplicated promot er region derived from subterranean clover stunt virus genome segment 7 in count er clockwise direction (Boevink, Chu et al. 1995)

P35S3 sequence including the promoter region from Cauliflower Mosaic Virus 35S transcript in clockwise dir ection (Odell, Nagy et al. 1985)

bar ; the coding sequence of the phosphinot hricin acetyltransferase gene of Streptomyces hygroscopicus in clockwise direction (De Block, Botterman et al. 1987)
3 ́nos sequence including the 3’ untranslated re gion of the nopaline synthase gene from the T-DNA of pTiT37 in clockwise direc tion (Depicker, Stachel et al. 1982) Left border (LB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988)



### C. Plant Pest Risk Assessment

Potential impacts to be addressed in this risk a ssessment are those that pertain to the use of BCS TwinLinkTM cotton and its progeny in the absence of confinement. APHIS utilizes data and information submitted by the applicant, in additio n to current literature, to determine if BCS TwinLinkTM cotton 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 that6 organism.

Based on the information requested by APHIS for submission of a petition for determination of nonregulated status (§ 340.6(c )) and in order to determine whether BCS TwinLinkTM cotton is unlikely to pose a plant pest risk, BRS consid ered information such as: plant pest risk characteristics, disease and pest susceptibilities, expres sion of the gene product, new enzymes, or changes to plant metabolism, weediness of the re gulated article, any impacts on the weediness of any other plant with which it can interbreed for BCS TwinLinkTM cotton. Issues related to agricultural or cultiva tion practices and the e ffects of the regulate d article on non-target organisms are considered in the Environm ental Assessment for BCS TwinLinkTM cotton.

Based on information on the biology of cotton (OECD 2008) data presented by BCS (Bayer CropScience 2010, APHIS Number 08 -340-01p) and scientific data relevant to a discussion of plant pest risk, APHIS co ncluded the following regard ing BCS TwinLinkTM cotton:



### 1. Potential impacts of altered disease and pest susceptibilities

APHIS assessed whether BCS TwinLinkTM cotton is likely to have sign ificantly increased disease and pest susceptibility. The assessm ent encompasses a thorough consideration of

introduced traits and interacti ons with pest and disease.

Cotton ( Gossypium hirsutum ) is not a plant pest in United States, and the introduced DNA in BCS TwinLinkTM cotton is unlikely to pose a plan t pest risk because there are no pathogenic DNA sequences present. The description of the genetic modifications, including genetic elements, expression of the gene product a nd their functions for BCS TwinLinkTM cotton has been summarized above. Because the goal of TwinLinkTM cotton was to decrease the pest susceptibility, the data submitted by BCS indi cated significant differences between BCS TwinLinkTM cotton and the non-transg enic counterparts for insect stresses (as measured by 32 agronomic parameters evaluated found on page 68 of Bayer CropScience 2010). An increase in yield was attributed to the inserted gene e xpression in TwinLinkTM cotton. There were no increases in pest susceptibility or disease as measured by the agronomic properties found on page 68 of the petition (Bay er CropScience 2010).

The introduced genes ( cry1Ab

, cry2Ae and bar ) encode for Cry1Ab and Cry2Ae and glufosinate synthase proteins, respectively. The cry1Ab and cry2Ae genes come from the soil bacteria Bacillus thuringiensis and the bar gene is from Streptomyces hygroscopicus . Such genes have been used in field trials previously and are not known to cause plant disease. There is no indication that inserting the cry1Ab

, cry2Ae and bar genes will result in increased likelihood of introduction or dissemination of a plant pest. Cry proteins were isolated and used commercially as a pesticide in France in 1938, marketed unde r the name Sporine. Since 1961, EPA approved Bt proteins for the treatment against lepidopteran pests under th e trade name, Thuricide (UCSD 1999; Erlandson and Litowski 2005). APHIS has not identified any plant pest risk following the introduction of Cry proteins or phosphinothricin acetyltransferase in products determined by APHIS to be no longer subject to the regulatory requirements of 7 CFR Part 340 or the plant pest provisions of the Plant Protection Act, such as rice, corn, cotton, beet, rapeseed, or soybean. A list of petition requests for a dete rmination of nonregulated status that have been approved by

APHIS can be found at http://www.aphis.usda.gov/brs/not_reg.html . The Plant Biosafety Office7 (PBO) of the Canadian Food Inspec tion Agency (CFIA) also concl uded that the intended effects herbicide-resistant plants such as phosphinothricin acetyltransferase are not related to altered plant pest potential ( http://www.inspection.gc.ca/english/ sci/biotech/enviro/herbice.shtml . CFIA has also reviewed the plant pest risk, the envi ronmental risk and safety of plants with novel genes containing Cry proteins a nd found these proteins are not re lated to altered plant pest potential. CFIA’s decision documents can be found at http://www.inspection.gc.ca/en glish/plaveg/bio/dde.shtml

.



### 2. Potential impacts from outcrossing of the BCS TwinLinkTM cotton to wild

relatives

In assessing the risk of gene introgressi on from BCS’ TwinLinkTM cotton into its sexually compatible relatives, APHIS considers two prim ary issues: 1) the potential for gene flow and introgression via polle n movement and horizontal gene transfer 2 ; and 2) the potential plant pest risk of introgression.



### a) Gene Flow via Pollen Movement

Movement of genetic material by pollen is possible only to those plants with the proper chromosomal type. In the United States, this would only include G. hirsutum

, G. barbadense

, and G. tomentosum . Native G. barbadense is only found in Hawaii, Vi rgin Islands and Puerto Rico, while G. tomentosum is only found in Hawaii (Fryxell 1979). G. hirsutum is generally self-pollinating but some cross- pollination can occur, albeit at relatively low incidence through activity of pollinating insects (Fr yxell 1979). Gene movement between G. hirsutum and G.

barbadense is possible if suitable insect pollinators ar e present, and if there is a short distancefrom host plants to recipient plants (Fryxell 1979). Physical barriers, intermediate pollinatorattractive plants, and other tem poral or biological impediment s (geography or absence of pollinators) reduce the potential for pollen m ovement (Fryxell 1979). Table 1 outlines the compatibility of all species on an international le vel. Concentration of suitable pollinators varies from location to location and by season, and is considerably suppressed by insecticide use.

Table 1. Cotton Species

Species Common Name Native

location Comments

G. hirsutum Upland cotton Central America, Mexico,

Caribbean

and

southern

Florida. Commercial Species, Grown in U.S.A. and comprises 97% of U.S.A cotton crop. Sexually compatible with G. barbadense

and G. tomentosum

.

G. barbadense Pima, Creole, Egyptian or Sea

Island cotton S. America Commercial species, grown in U.S.A. Grown in Hawaii, Virgin Islands and Puerto Rico. Sexually compatible with G.

hirsutum and G. tomentosum

.

G. tomentosum Ma’o or Hawaiian

cotton Hawaii Non-commercial species . Thought to be only pollinated by moths when the flowers

open at night, but more recent evidence

2 Horizontal gene transfer is any process in which an organi sm transfers genetic material to another cell that is not its offspring.8 suggests it can also be pollinated in early morning by bees. Only found in Hawaii. Sexually compatible with G. hirsutum and

G. barbadense

.

G. arboreum Asiatic tree or tree

cotton Pakistan,

India Commercial species, grown in Europe, Africa and eastern countries. Sexually compatible with G. herbaceum

.

G. herbaceum Levant cotton Africa,

Arabia Commercial species, grown in Europe, Africa and eastern countries. Sexually compatible with G. arboreum

.

G. thurberi Thurber’s, Desert or

Arizona desert

cotton Mexico,

Arizona Non-commercial species. Sexually compatible with G. arboreum and G.

herbaceum

.

Historically, it was reported that cross-pollination between G. tomentosum and G. hirsutum was unlikely because they use different insect pollinat ors and are receptive to pollination at different times of the day (McGregor 1976). Field and labora tory studies demonstrat ed that the historic literature is incorrect, and that these three sp ecies share common pollinators, and further that

differences in flower structure a nd flowering habits do not serve as barriers to cross pollination (Pleasants and Wendel 2010). However, DNA marker analyses have not found evidence of genes

from G hirsutum occurring in native populations of G. tomentosum (DeJoode and Wendel 1992). It is possible that the lack of evidence of movement of G. hirsutum genes into G. tomentosum is the result of lack of opportunity because cotton has not been gr own commercially in Hawaii for

at least the last 45 years (USDA-APHIS 2010).

In farm scale studies using traditional Upland cotton in California, it was found that the outcrossing distance was strongly dependent on the presence of bee colonies. When only native pollinators were present in the field, 1% out-cross ing was detectable over a distance of 1 meter (approximately 3 ft) and 9 m (29.5 ft) when th ere was high pollinator activity (Van Deynze, Sunderstrom et al. 2005). Out- crossing declined exponentially w ith increasing distance from the source plot (Van Deynze, Sunderstrom et al. 2005) . Current cultivation practices to prevent outcrossing (distance being primarily used) have been deemed sufficient to prevent unwanted gene flow. For Upland cotton, the Association of Official Seed Certifying Agencies (AOSCA) mandates an isolation distance being a nature ba rrier or crop boundary with a minimal isolation distance of 100 ft “if the contaminating s ource differs by easily observed morphological characteristics from the field to be inspected”. For Pima or Egyptian t ype cotton “the isolation shall be 1320 feet from any othe r type of cotton for Foundation a nd Registered and 660 feet for

Certified seed” 3 . Since TwinLinkTM cotton is not mo rphologically distinguishable from traditional Upland cotton much like Pima or E gyptian type cotton, cultiv ation practices using

AOSCA standards of 1320 ft for Foundation and Re gistered and 660 ft for Certified seed are

used.

Wind is rarely seen as a mean s for cross-pollination of cotton pollen because of its adherent properties and large size (mean diameter of 53-56  m). The pollen of cultivated Gossypium species is described as being sticky and havi ng pronounced spines, with a marked tendency for groups of pollen grains to clump toge ther (Hutmacher and Wright 2006).

3 From AOSCA “Yellow Books” 2003 OPERATIONAL PROCEDURES, CROP STANDARDS AND SERVICE

PROGRAMS PUBLICATION (Genetic and Crop Standards), pg 194.9 

### b) Gene Flow via Horizontal Gene Transfer 4

Transfer and expression of DNA from TwinLinkTM cotton to soil bact eria is unlikely to occur. Gebhard and Smalla (Gebhard and Smalla 1999) and Schlüter et al. (Schlüter, Fütterer et al. 1995) have studied transgenic DNA movement to b acteria, and although theo retically possible, determined mathematically it would occur at extremely low rates (approximately 1 in 10 -14 ). Many genomes (or parts thereof) have been sequen ced from bacteria that are closely associated with plants including Agrobacterium and Rhizobium (Kaneko, Nakamura et al. 2000) and there is

no evidence for recent horizontal transfer. Koonin et al. (Koonin, Makarova et al. 2001) and Brown (Brown 2003) presented reviews based on se quencing data that revealed horizontal gene transfer occurs occasionally on an evolutionary time scale of millions of years. Even in the unlikely event transfer were to occur, the ge ne would be poorly expr essed at best because transgene promoters and coding sequences are optimized for plant expression and function poorly in prokaryotic cells.

Based on the above considerations, TwinLinkTM cotton will not adversely impact sexually compatible wild relatives or their weediness characters.



### 3. Potential impacts based on the relative weediness of BCS TwinLinkTM

cotton.

In the United States, cotton is not listed as a weed in the major weed references (Crockett 1977; Holm, Pancho et al. 1979; Muenscher 1980), nor is it present on the list of noxious weed species (7 CFR 360) distributed by the Federa l Government (USDA-APHIS 2006). Furthermore, cotton has been grown throughout the world without any report that it is a serious weed. Cotton is unlikely to become a weed. It is not pers istent in undisturbed

environments without human intervention. In the year following cultivation, cotton may grow as a volunteer only under specific conditions (disturbed or cultivated soil that had cotton grown in the last growing season) and can be easily controlle d by herbicides or mechanical means. It does not compete effectively with cultivated plants or primary colonizers beca use it is such a slow grower, especially in the cooler soils in the norther n cotton belt (OECD 2004).

APHIS assessed whether TwinLinkTM cotton is any more likely to become a weed than the isogenic nontransgenic cotton line, or other cott on varieties currently under cultivation. The assessment encompasses a thorough considera tion of the basic biology of cotton and an evaluation of the unique characteristics of Twin LinkTM cotton evaluated under field conditions.

BCS conducted agronomic field trials during the 2005, 2007 and 2008 growing seasons across 8 states and 23 locations representative of the ma jor cotton-growing areas of the cotton belt (field test reports can be found in Bayer CropScien ce 2010 in Appendix 1, p.102). The data submitted by BCS indicated no significant difference s between BCS TwinLinkTM cotton and the nontransgenic counterparts (as meas ured by 32 agronomic parameters evaluated found on page 68 of Bayer CropScience 2010).

4 Horizontal gene transfer is any process in which an organi sm transfers genetic material to another cell that is not its offspring.10 Based on the agronomic field data and literat ure survey about cott on weediness potential, TwinLinkTM cotton lacks ability to persist as tr oublesome weed, and there would be no direct impact on current weed management practices for cotton cultivation.



### 4. Potential impacts on target and nontarget organisms, including

beneficial organisms.

The Cry1Ab and Cry2Ae proteins expressed in TwinLinkTM cotton are effective in controlling specific lepidopteran larvae such as bollworm (CBW, Helicoverpa zea ), tobacco budworm (TBW, Heliothis virenscens ) larvae and fall armyworm (FAW, Spodoptera frugiperda ) which are common pests of cotton. These Cry proteins ar e not expected to adversely affect non-target invertebrates such as bees, ladybugs, lacewings, spiders or other arthropods and vertebrate organisms, including birds, mammals and humans, because they do not contain the receptor found in the midgut of target insects (OECD 2007).

Cry1Ab and Cry2Ae are crys tal delta-endot oxins from Bacillus thuringiensis . A total of 314

different cry genes have been identified by 2005 (OECD 2007). The insectic idal activity of Cry1Ab and Cry2Ae proteins occurs when the C-te rminal half of these inactive protoxins are enzymatically cleaved within the midgut of suscep tible insect larvae by trypsin-like proteases to the active toxin, which consists of the N-terminal portion of the molecule (Federici 1993; Brar, Verma et al. 2007). The susceptibility of the inse ct depends on what trypsin-like protease is expressed in the specific insect’s gut (OECD 2007).

For many decades microbial products containing Bacillus thuringiensis (the organism that produces Cry proteins) have been used to cont rol insect pests on a commercial scale and for home garden applications (Glare and O’Callaghan 2000; Shelton, Zh ao et al. 2002). Plants that were genetically engineered to express the Cry1A protein have a history of safe use in the U.S. Since the mid-1990s, corn and cotton lines have been commercialized without substantiated reports of significant deleterious impacts on non-target organisms (OECD 2007; USEPA 2008).

As stated on pp.36 of the submitted petition (Bayer CropScience 2010) “Over the past 50 years,

current use of Bt pesticides, including those expressing Cry1 Ab, is estimated to be several tons annually. Moreover, Cry1Ab protein is expressed in a number of genetically modified crops that have been approved since 1995 and are currently commercialized. No records of allergenicity in

humans and mammals were found associated with Bt bacteria (OECD, 2007). In addition,

microbial Bt biopesticides, including thos e containing the Cry1Ab protein, have shown no toxic

effects in several mammalia n toxicity studies (Betz et al ., 2000).”

Cry proteins are not exp ected to adversely affect non-target invertebrates, such as bees, and vertebrate organisms, including birds, mammal s and humans, because they do not contain the receptor found in the midgut of target insects. Data provided in the petition (Bayer CropScience 2010, summarized in the Table 30 and 31, pg. 80-81 and data found on pg. 82-83) confirmed that in the mammal, bird, honey bee, above ground ar thropod, and soil dwelling invertebrate studies,

no observable adverse effects or differences in surv ival were noted at for both Cry proteins. The amount of the Cry proteins tested were well a bove those expected from exposure to the Cry proteins from TwinLinkTM cotton planted in th e field. The nontarget above-ground arthropods and soil-dwelling invertebrates studied (la dybugs, green lacewings, springtail, daphnia,11 honeybees, collembola and earthworms) were consider ed to be representativ e of the cotton agroecosystem.

Although not an endangered or threatened species, Danaus plexippus (monarch butterfly) is a species of high conservation interest, and ther e has been concern that it may be harmed by consuming pollen from transgenic insect-protect ed cotton and corn. The monarch is susceptible to Cry1Ab (Hellmich, Siegfried et al. 2001) the most common insecticidal protein in transgenic maize and cotton. However, the distribution of the monarch’s food plant ( Asclepias syriaca

- common milkweed), the monarch’s pattern of migration (Sears, Hellmich et al. 2001), and the lack of cotton pollen movement and adherence to available milkweed (cotton pollen is heavy and sticky) means that very few monarchs are e xposed to harmful concentrations of Cry1Ab. TwinLinkTM cotton, therefore, poses low risk to monarchs because of mi nimal hazard of Cry1Ab and Cry2ac and low exposure to Cry protein-co ntaining pollen. TwinLinkTM cotton is expected to have no harmful effects on any endangere d or threatened species in the U.S.



### 5. Potential impacts from transferring genetic information from BCS

TwinLinkTM cotton to organisms with which it cannot interbreed.

APHIS examined the potential for the new genetic material inserted into TwinLinkTM cotton to be horizontally transferred to other organisms without sexual reproduction and whether such an event could lead directly or indi rectly to disease, damage, injury or harm to plants, including the creation of more virulent pathogens. The hor izontal gene transfer between unrelated organisms is one of the most intensively stud ied fields in the biosciences since 1940, and the issue gained extra attention with the release of transgenic plants into the environment (Dröge, Puhler et al. 1998). Potential risk s from stable horizontal gene tr ansfer (HGT) from genetically engineered organisms to another organism wit hout reproduction or human intervention were recently reviewed (Keese 2008). Mechanisms of HGT include conjugation, transformation and transduction, and other diverse mechanisms of DNA and RNA uptake and recombination and rearrangement, most notably through viruses a nd mobile genetic elements. HGT has been a major contributor to the spread of antibiotic resistance amongst pathogenic bacteria and the emergence of increased virulence in bacteria, eukaryotes and viruses and in the long run has contributed to major transitions in evolution.



### a) Potential for Horizontal Gene Transfer to Bacteria or Fungi

TwinLinkTM cotton has three bacteria genes. Hori zontal gene transfer and expression of DNA from a plant species to other bacterial specie s is unlikely to occur based on the following observations. Although there are many opportunities for plants to directly in teract with fungi and bacteria (e.g. as commensals, sy mbionts, parasites, pathogens, d ecomposers, or in the guts of herbivores), there are almost no evolutionary exam ples of HGT to bacteria from eukaryotes or from plants to fungi (Keese 2008). The only genes likely to be transferred successfully from genetically engineered plants to bacteria are other bacterial genes. Horizontal transfer from and expression in bacteria of the foreign DNA in serted into the nucl ear genome of TwinLinkTM cotton is unlikely to occur. First, many genomes (or parts thereof) have been sequenced from bacteria that are closely asso ciated with plants including Agrobacterium and Rhizobium (Kaneko, Nakamura et al. 2000; Wood, Setubal et al. 2001). There is no evidence that these organisms contain genes derived from plants. Second, in cases where review of sequence data12 implied that horizontal gene transfer occurr ed, 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). Third, transgene DNA promoters and c oding sequences are optimized for plant expression, not prokaryotic bacteria l expression. Thus even if horiz ontal gene transfer occurred, proteins corresponding to the transgenes are no t likely to be produced. Fourth, the FDA has evaluated horizontal gene transfer from the use of antibiotic resistance marker genes and

concluded that the likelihood of transfer of antibiotic resistan ce genes from plant genomes to microorganisms in the gastrointes tinal tract of humans or animals, or in the environment, is remote (http://vm.cfsan.fda.gov/~dms/opa-armg.html).



### b) Potential for Horizontal Gene Transfer to Viruses

APHIS also considered whethe r horizontal transfer of DNA from TwinLinkTM cotton to plant viruses was likely to occur and would lead to the creation or selection of a more virulent plant pathogen through recombination with other plant viru ses. This issue has b een considered before by other science review panels and government regulatory bodies (Keese 2008). The only virus sequences contained within TwinLinkTM cotton en code regulatory elements: the cauliflower mosaic virus, the cassava vein mosaic virus a nd the subterranean clover stunt virus. Regulatory elements such as promoters and terminators have not been implicated in viral recombination.

Therefore, APHIS concludes that horizontal gene transfer is un likely to occur and thus poses no significant environmental or plant pest risk. Finally, under natural conditions; no tr ansfer of an intact functional gene has been demonstrat ed to date (Miki and McHugh 2004). Therefore

APHIS concludes that horizontal gene transfer is unlikely to o ccur and thus poses no significant environmental or plant pest risk.



### D. Conclusion

APHIS prepared this plant pest risk assessment in order to determine if BCS TwinLinkTM cotton is unlikely to pose a plant pest risk. Due to the l ack of plant pest risk from the inserted genetic material, the lack of weediness characteristic s of BCS TwinLinkTM cotton , the lack of atypical responses to disease or plant pe sts in the field, the lack of de leterious effects on non-targets or beneficial organisms in the agro-ecosystem, and th e lack of horizontal ge ne transfer, APHIS has

concluded that BCS TwinLinkTM cotton is unlikely to pose a plant pest risk.



### E. References

Bayer CropScience (2010). Petition for the determ ination of nonregulated status for insectresistant and glufosinate ammonium-tolerant cotton; TwinLinkTM cott on (events T304-40 and GHB119). Submitted by A. L. Scott, Regulator y Affairs Manager. Bayer CropScience LP (See Table http://www.aphis.usda.gov/ biotechnology/not_reg.html ). Boevink, P., P. W. G. Chu, et al. (1995). "Se quence of subterranean clover stunt virus DNA: Affinities with the geminiviruses." Virology 207 : 354-361. Brar, S. K., M. Verma, et al. (2007). "Bacillus thuringiensis proteases : Production and role in growth, sporulation and synergism." Process Biochemistry 42 (5): 773-790. Brown, J. R. (2003). "Ancient hori zontal gene transfer." Genetics 4 : 121-132.13 Crockett, L. (1977). Wildly successful plants : North American weeds. Honolulu, Hawaii, University of Hawaii. De Almeida, E. R. P., V. Gosselé, et al. ( 1989). "Transgenic expressi on of two marker genes under the control of an Arabidopsis rbcs prom oter: sequence encodi ng the Rubisco transit peptide increase expression leve ls." Molec. Gen. Genet. 218 : 78-86. De Block, M., J. Botterman, et al. (1987). "E ngineering herbicide resistance in plants by expression of a detoxifying enzyme." EMBO Journal 6 : 2513-2518. DeJoode, D. R. and J. F. Wendel (1992). "Genetic diversity and origin of the Hawaiian Islands

cotton, Gossypium tomentosum ." American Journal of Botany 79 (11): 1311-1319. Depicker, A., S. Stachel, et al. (1982). "N opaline Synthase: transcript mapping and DNA sequence." J. Molec. Appl. Genet. 1 : 561-573. Dröge, M., A. Puhler, et al. (1998) . "Horizontal gene transfer as a biosafety issue: A natural phenomenon of public concern." Journal of Biotechnology 64 : 75-90. Erlandson, M. and T. Litowski. (2005). "What is Bt?" Retrieved January 7, 2010, from http://www.odec.ca/projects/2005/ erla5m0/public_html/bib.html#12

. Federici, B. A. (1993). "Insecticid al bacterial proteins identify the midgut epithelium as a source of novel target sites for insect control." Ar chives of insect bioc hemistry and physiology

22 : 357-371. Fryxell, P. A. (1979). The natura l history of th e cotton tribe ( Malvaceae , tribe Gossypieae ). College Station and London, Texas A&M University Press, Gebhard, F. and K. Smalla (1999). "Monitoring field releases of ge netically modified sugar beets for persistence of transgenic plant DNA and horizontal gene transfer." FEMS Microbiology Ecology 28 : 261-272. Glare, T. R. and M. O’Callaghan (2000). Bacillus thuringiensis Biology, Ecology and Safety. Chichester, John Wiley & Sons, pp.368. Harpster, M. H., J. A. Townse nd, et al. (1988). "Relative strengths of the 35S Cauliflower Mosaic Virus, 1’, 2’ and nopaline synthase promoters in transformed tobacco, sugarbeet and oilseed rape callus tissue." Molec. Gen Genet. 212 (182-190). Hellmich, R. L., B. D. Siegfried, et al . (2001). "Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen." Proceed ings of the National Academy of Sciences (USA) 98 : 11925-11930. Höfte, H., H. de Greve, et al. (1986). "Structural and functional analysis of a cloned delta

endotoxin of Bacillus thuringiensis berliner 1715." Eur. J. Biochem. 161 : 273-280. Holm, L., J. V. Pancho, et al. (1979). A geographical atlas of wo rld weeds. NY, John Wiley and Sons, pp.391. Hutmacher, R. B. and S. D. Wright. (2006). "Methods to enable the coexis tence of diverse cotton production systems, Publication 8191." Agricult ural Biotechnology in California Series. Kaneko, T., Y. Nakamura, et al. (2000). "Com plete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti ." DNA Research 7 (6): 331-338. Keese, P. (2008). "Review Article: Risks from GMOs due to horizontal gene transfer." Environmental Biosafety Research 7 (123-149). Koonin, E. V., K. S. Makarova, et al. (2001). "Horizontal gene transfer in prokaryotes: Quantification and classification." Annual Review of Microbiology 55 : 709-742. Marshall, J. S., J. D. Stubbs, et al. (1996). "T wo genes encode highly similar choroplastic NADP-malic enzymes in Flaveria." Plant Physiol. 111 : 1251-1261.14 McGregor, S. E. (1976). Insect pollination of cultivated crop plants. Agricultural Handbook No 496. Washington, DC, USDA-ARS. Michiels F., M. S., Scheirlinck T., Komari T. (1992). Stamen-specific promoters from rice.

PLANT GENETIC SYSTEMS N.V. BE. Miki, B. and S. McHugh (2004). "Selectable marker genes in transgenic plants: applications,

alternatives and biosafet y." Journal of Biotechnology 107 : 193-232. Muenscher, W. C. (1980). Weeds, 2nd Edition. Ithaca and London, Cornell University Press, Odell, J. T., F. Nagy, et al. (1985) . "Identification of DNA sequences required for activity of the

Cauliflower Mosaic Virus 35S promoter." Nature 313 : 810-812. OECD (2004). "Consensus Document on compositional considerations for new varieties of cotton ( Gossypium hirsutum and Gossypium barbadense ): key food and feed nutrients

and anti-nutrients." ENV/JM/MONO(2004)16

. OECD (2007). Consensus document on safety in formation on transgenic plants expressing Bacillus thuringiensis -derived insect control protein : 109 OECD (2008). Consensus Document on the Biol ogy of Cotton (Gossypium spp.). OECD Series on Harmonisation of Regulatory Oversi ght in Biotechnology Paris, OECD. ENV/JM/MONO(2008)38 No. 45: 64. Pleasants, J. M. and J. F. Wendel (2010). "R eproductive and pollination biology of the endemic Hawaiian cotton, Gossypium tomentosum (Malvaceae)." Pacific Science 64 (1): 45-55. Sanfaçon, H., P. Brodman, et al . (1991). "A dissection of th e Cauliflower Mosaic Virus polyadenylation signal." Genes and Development 5 : 141-149. Schlüter, K., J. Fütterer, et al. (1995). "Horizontal gene transfer fr om a transgenic potato line to a bacterial pathogen ( Erwinia chrysanthemi ) occurs if at all at an extremely low frequency." Nature Biotechnology 13 : 1094-1098. Sears, M. K., R. L. Hellmich, et al. (2001). " Impact of Bt corn pollen on monarch butterfly populations: A risk assessment." Proceedings of the National Academy of Sciences (USA) 98 : 11937-11942. Shelton, A. M., J.-H. Zhao, et al. (2002). "E conomic, ecological, food safety, and social consequences of the deployment of Bt tran sgenic plants." Annual Review of Entomology

47 : 845-881. UCSD. (1999). "History of Bt." Retrieved January 7, 2010, from http://www.bt.ucsd.edu/

. USDA-APHIS. (2006). "Plant Heal th: Noxious Weeds Program." Retrieved 08/22/2007, from http://www.aphis.usda.gov/plant_hea lth/plant_pest_info/weeds/index.shtml

. USDA-APHIS (2010). Draft Environmental Asse ssment, Syngenta Biotechnology, Inc. Petition (07-108-01p) for Determination of Nonregulated Status for Lepidopteran-Resistant Event COT67B Cotton. Riverdale, MD , U.S. Department of Agriculture Animal and Plant Health Inspection Service. USEPA (2008). Bacillus thuringiensis modified Cry1Ab (SYNIR67B-1) and Vip3Aa19 (SYNIR102-7) insecticidal proteins and the genetic material necessary for their production in COT102 X COT67B cotton. Washington, D.C., United States Environmental Protection Agency http://www.epa.gov/pesticides/biopestic ides/ingredients/tech_docs/brad_006529.pdf

. Van Deynze, A. E., F. J. Sunderstrom, et al. (2005 ). "Pollen-mediated gene flow in California cotton depends on pollinator activity." Crop Science 45 : 1565-1570.15 Verdaguer, B., A. De Kochko, et al. (1996). "Iso lation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic vi rus (CVMV) promoter." Plant Mol. Biol. 31 : 1129-1139. Wood, D. W., J. C. Setubal, et al. (2001). "T he genome of the natural genetic engineer Agrobacterium tumefaciens C58." Science 294 : 2317-2323. Zambryski, P. (1988). "Basic processes underl ying Agrobacterium-mediated DNA transfer to plant cells." Ann. Rev. Genet. 22 : 1-30.