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## Plant Pes **t Risk Assessment for Event 5307 Corn**  Sy ngenta Biotechnology , Inc. has petitioned APHIS (APHI S number 10 - 336 - 01p ) for a determination that genetically engineered (GE) corn (Zea mays L. subsp. mays ) Event 5307 is unlikely

to pose a plant pest risk (Sy ngenta 2011) and, therefore, should no longer be a regulated article under APHI S’ 7 Code of Federal Regulations (CFR) part 340. APHI S administers 7 CFR part 340 under the authority of the plant pest provisions of the Plant Protection Act of 2000 1

. This plant pest risk assessment was conducted to determine whether corn Event 5307 is unlikely to pose a plant pest risk under full deregulation. Event 5307 was produced by transformation of an immature corn embryo of li ne NP222 with Agrobacterium tumefaciens. Because A. tumefaciens is a plant pest and some of the regulatory

sequences (promoter from Cestrum yell ow leaf curling virus and terminator from A. tumefaciens ) used to facilitate expression of the genes in corn were derived from plants pests, this corn has been considered a regulated article under APHIS regulations at 7 CFR part 340. Potential impacts to be addressed in this risk assessment are those that pertain to the use of Event 5307 and its progeny in the absence of confinement. APHI S utilizes data and information submitted by the applicant, in addition to current literature, to determine if Event 5307 is unlikely

to pose a plant pest risk. If APHI S determines that a GE organism is n ot a plant pest risk , then APHI S has no regulatory authority over that organis m. Potential impacts in this Plant Pest 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 enzy mes, 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. The Environmental Assessment (EA) for thi s petition considers whether agricultural or cultivation practices for Event 5307 may result in impacts on the environment. A thorough assessment of the effects of the determination on non - target and beneficial organisms, and threatened and endangered spec ies is included in the EA . **Development of Event 5307 Corn** Corn is the most widely

produced feed grain in the U.S., accounting for more than 90

1 Section 403 (14) of the Plant Protecti on 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.”2

percent of the total value and production of feed grain (USDA - ERS 2011). There is a need to provide growers with a cost effective method to control insects, while taking into account the potential haz ardous nature of insecticides. GE corn expressing Cry proteins derived from Bacillus thuringiensis ( Bt), commonly

referred to as Bt corn , have a long history of safe use without adverse human health or environmental effects and provide an option for the control of coleopteran insect pests (NAS 2004). The Bt proteins used in this product called eCry3.1Ab controls corn rootworm, which is a highl y destructive pest respons ible for the single largest use o f conventional insecticides in the United States (EPA 2007a). Corn rootworms have long been the major economic pests of corn in the U.S. (Calvin 2003 ). Sy ngenta h as genetically

engineered corn to express an eCry3.1Ab protei n for use in the control of coleopteran pests. Event 5307 deters feeding b y s everal insects known to cause significant damage to corn in the U.S. These include: western corn rootworm ( Diabrotica vergifera virgifera Le Conte), northern corn rootworm ( D. longicornis barberi Smi th and Lawrence ) and Mexican corn rootworm ( D. virgifera zeae Kry san and Smith) (Peairs and Pilcher 2010) . GE corn containing insect - resistant traits, first appro ved in the U.S. in 1996, has been wide ly adopted by

U.S. growers (Figure1) . Adoption of stacked varieties (those containing both herbicide and insect tolerance traits) has accelerated in recent years. Planting of stacked corn made up 49 percent of corn acres in 2011 (Figure 2).

Figure 1. Percent of acreage of genetic all y engineered crops in the U.S., 1996 - 2011 (USDA ERS 2011 ).3

Figure 2. Percent of planted acres of corn in the U.S. by trait, 2000 - 2011 (USDA - ERS 2011). The eCry3.1Ab protein is a chimeric Bacillus thuringiensis protein, composed of portions of Cry 1Ab and mCry3A proteins both of which are EPA - registered Plant Incorporated Protectants (75 FR 34040 - 34045). O n June 16, 2010 EPA issued a temporary exemption from the requir ement of tolerance for eCry3.1Ab protein in co rn that expires June 1, 2012 (EPA 2010 ). Sy ngenta filed for registration of pesticidal use of the eCry3.1Ab protein in corn on January 21, 2011 (EPA 2011). Phosphomannose isomerase (PMI), the selectable marker protein produced by Event 5307 corn plants, is exempt fro m food and feed tolerances (EPA 2007 b ). Sy ngenta initiated a voluntary

pre - market consultation process with FDA and submitted a safety and nutritional assessment for Event 5307 corn in January 2011 (File BNF 000128). Description of the Modification Event 5307 corn was produced b y transformation using disarmed A. tumefaciens ( Sy ngenta 2011, pp. 26 - 28, Figure s II I - 1, III - 2). Immature embryos of Sy ngenta inbred corn line NP 2222 (Plant Variety Protection C ertificate 2 00200071; US DA - AMS 2004) were infected with A. tumefaciens strain LBA440 4 containing plasmid SYN12274. Plants containing the introduced DNA were selected based on survival on a mannose substrate d ue to the expression of phospho mannose isomerase (S yn genta 2011, p . 27, Figure III - 2). The plasmid SYN12274 contained two gene expression cassettes flanked by the right and left border of T - DNA from the A. tumefaciens Ti - plasmid ( Sy ngenta 2011, pp. 28, 35 - 36, Figure III - 1, Table IV - 1). eCry3.1 Ab

– expression cassette consists of three genetic elements (S yn genta 2011, Figure III - 1, Table IV - 1).4

 The Cestrum Yellow Leaf Curling Virus (CmYLCV, Caulimoviridae dsDNA plant virus 2 ) genomic full - length transcript promoter (Hohn 2007; Stavone 2003 ).

 The ecry3.1Ab gene consists of a fusion between the 5’ end (Domain I, Domain II and 15 amino acids of Domain I II) of a modified mcry3A gene from B. thuringiensis subsp. tenebrionis and the 3’end (Domain III and variable region 6) of a cry1Ab (Sy

ngenta 2011 , Table IV - 1, Figure IV - 1 ; Walters 2010 ).

 The nopaline s ynthase (NOS) terminator from A. tumefaciens (Depicker 1982). Phosphomannose isomerase

– expression cassette consists of three genetic elements ( Sy ngenta 2011, Figure III - 1, Table IV - 1).

 The poly

ubiquitin promoter from Z. mays (Christensen 1992).

 The pmi gene from Escherichia coli strain K- 12 (Negrotto 2000).

 The NOS terminator from A . tumefaciens (Depicker 1982). Data from Southern blot anal ys is was provided and reviewed by APHI S that showed that Event 5307 contains: (1) a single cop y of the CmYL CV promoter (S yn genta 2011, Figure s V - 4, V - 5, Table V - 2); (2 ) a single cop y of the ecry3.1Ab gene (S yn genta 2011, Figure s V - 2, V - 3, Table V - 1); (3) two copies of the NOS terminator ( Sy ngenta 201 1, Figure s V - 10, V - 11, Table V - 5); and (4) a single copy of the pmi gene (Sy

ngenta 2011, Figure s V - 6, V - 7, Table V - 3). Southern blot anal ysis demonstrated the absence of the plasmid backbone elements ( Sy ngenta 2011, Figure s V - 12, V - 13, Table V - 6). Data fro m Southern anal ys is was provided and revie wed b y APHI S that demonstrates st able integration and inheritance of the 5307 corn insert over four generation (Syngenta 2011, Figure s V - 14, V - 15, V - 16, Table V - 7). **Potential of 5307 to Become Invasive and/or a Weed**  Corn possess es few of the characteristics of those plants that are notabl y successful as weeds (Baker 1965; Keeler 1989). In the U.S., corn is not listed as a weed (Crockett 1977; Holm 1991 ; Muenscher 1980), nor is it present in the Federal noxious we ed list (7 CFR part 360 3 ). Furthermore, corn is grown throughout the world without any report that it is a serious weed or that it forms persistent feral populations (Gould 1968). Like many domesticated crops, corn seed from a previous ye ar’s crop can over winter and germinate the following ye ar. For instance, the appearance of corn seedlings in soy bean fields following a corn crop is a common occurrence. Manual or chemical measures are often applied to remove these volunteers, but the plants that are not removed do not ty picall y result in feral populations in following years.

2 http://ictvdb.cumc.columbia.edu/servlet/Virus?id=944 3 http://www.aphis.usda.gov/plant_health/ plant_pest_info/weeds/dow nloads/weedlist.pdf5

APHI S assessed whether Event 5307 is any more l ikely

to become a weed than a nearis ogenic non - transgenic corn line. The assessment encompasses a thorough consideration of the basic bio logy

of corn and an evaluation of the unique characteristics of Event 5307 (Sy ngenta 201 1 Table VI I - 1 , Appendix A Table A - 1 ). Sy ngenta has been conducting agronomic field trials of Event 5307 since 2005 at locations that provide a range of environmental co nditions representative of where Event 5307 is expected to be grown (Sy ngenta 2011, Appendix A, Table A - 1) . Field trial data indicated that Event 5307 does not exhibit characteristics that would cause it to be weedier than the parental corn line (Sy ngenta 2011 Table s VII - 5, VII - 6, VI I - 7, VII - 8, VII - 9, VI I - 10) . No differences in phenoty pic characteristics that might contribute to enhanced weediness were observed between Event 5307 and control lines for the wide range of phenotypic endpoints assess ed in these field trials or in greenhouse or laboratory experiments (Syngenta 2011 , Table s VII - 7, VII - 8, VII - 9, VII - 10, VI - 12, Figure VII - 1 ). There was no increase in weediness potential as measured b y differences in seed germination, dormancy , plant emerg ence, plant height, ear height, stalk lodging, barren plants, timing of pollen shed or silking, pollen viability

and morp hology , and yi eld (Sy ngenta 2011 , Table s VII - 7, VII - 8, VII - 9, VII - 10, VI - 12, Figure VII - 1). None of the measure attributes showed any differences relative to its comparator, suggesting that Event 5307 is not weedier than current corn cultivars. These results on growth characteristics, seed production and germination indicate that Event 5307 is not significantly different from its comparat ors. There is no indication that Event 5307 possesses a selective advantage that would result in increased weediness. As with other corn varieties, Event 5307 lacks the ability to persist as a troublesome weed, and there would be no significant impact on current weed management practices for corn cultivation. **Potential for Gene Flow and Gene Introgression from Event 5307 into Sexually**   ** Compatible Relatives** Gene flow from crops to wild relatives may have the potential to enhance the weediness of wild relatives , as observed in rice, sorghum, sunflower, and a few other crops (Ellstrand 1999). Gene flow is a natural biological process with significant evolutionary importance. A number of angiosperm taxa are believed to be derived from hy bridizati on or introgression between closely related taxa (Soltis and Soltis 1993; Rieseberg 1997) and even in existing floras, the occurrence of hyb ridization or introgression is report ed to be widespread (Stace 1987; Rieseberg 1997 ) . It has been a common practice by plant breeders to artificiall y introgress traits from wild relatives into crop plants to develop new cultivars. APHI S evaluated the potential for gene introgression to occur from Event 5307 to sexually compatible wild relative s and considered whether s uch introgression would result in increase d weediness (Sy ngenta 2011, Figure III - 3, Table III - 1) . Cultivated corn is sexually

compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum (OECD 2003). The c losest wild relatives of corn, various Zea species referred to as teosinte, are normally confined to the tropical and6

subtropical regions of Mexico, Guatemala, and Nicaragua. In the U.S. a fairly rare, sparsel y dispersed feral population of teosinte has be en reported in Florida (USDA PLANTS D atabase 4 , accessed 8/23/2011 ). The genus Tripsacum contains up to 16 recognized species, most of which are native to Mexico, Central and South America. Three of these species ( T. dactyloides, T. floridatum, and T. lanceolatum ) exist as wild and/or cultivated species in the continental U.S.; and two taxa ( T. faciculatum and T. latifolium ) also occur in Puerto Rico (USDA PLANTS Database, accessed 8/23/2011). Though many of these species occur where corn might be grown , gene introgression from Event 5307 under natural conditions is highl y unlikely. Hybrids of Tripsacum species with Zea are difficult to obtain outside of a laboratory

and are often sterile or have greatly reduced fertility

and none of them ty picall y withs tand even the mildest winters (Galinat 1988; Ellstrand 2007). First generation hybrids are generall y less fit for survival and dissemination in the wild and show substantially

reduced reproductive capacity, which thus acts as a significant constraint to introgression. Therefore, USDA has concluded that adverse consequences of gene flow from Event 5307 to wild or weedy species in the U.S. are highl y unlikely . **Potential Impacts on Target and Non -**  **target Organisms, Including Beneficial Organisms**  Based on the data provided by the applicant and existing literature, APHI S evaluated the potential for plant pest - related impacts from Event 5307 on non - target or beneficial organisms. The genes introduced into Event 5307 result in the production of phosph omannose isomerase (PMI) and eCry3.1Ab proteins. The pmi gene, which was introduced along with the ecry3.1Ab gene via the same pSYN12274 transformation vector, was employed as a selectable marker during the process of regenerating plant material following transformation (S yn genta 2011, p. 40, Figure V - 1). The pmi gene was isolated from E. coli , which is a common intestinal bacterium and is considered a non - allergenic source of proteins (FAO/WHO 2001). The PMIprotein is widel y distributed in nature and has been found to be expressed in enteric bacteria, fungi, insects, some species of plants, and mammals (EPA 2004, Slein 1950). Additionally , EPA has granted an exemption from the requirement of a tolerance for the PMIprotein as an inert ingredient in all pl ants (EPA 2004). The ecry3.1Ab gene from Bacillus thuringiensis expressed in Event 5307 is a chimeric gene composed of portions of the N - terminus of mcry3A gene and the C - terminus of cry1Ab gene (S yn genta 2010, Figures IV - 1A, IV - 1B). For decades, microbial products containing B. thuringiensis have been used to control insect pests on a commercial scale and for home garden applications (Glare and O'Callaghan 2 000; OMRI 2011; Shelton 2002). Because Cry protein receptors are not present in non - target organisms (Glare and O'Callaghan 2000; Hofmann 1988a; Hofmann 1988b; Shimada 2006a; Shimada 2006b; Van Rie 1989; Van Rie 1990), these insecticidal proteins are not expected to adversel y

4 http://plants.usda.gov/java/county? state_name=Florida&statefips=12&symbol=ZEME7

affect non - target invertebrate and vertebrate organisms (Glare and O'Callaghan, 2000; EPA 2007a; EPA 2008). Since the commercialization of Bt crops, a large number of field studies published in the scientific literature have addressed the issue of long range effects of cultivation of C ry proteins on the invertebrate community structure where Bt crops are grown. The use of transgenic crops pro ducing Cry proteins has been shown to reduce the use of broad spectrum insecticides 5 without significant impacts on non - target organisms ( Cattan eo 2006; Dively

2005; Marvier, 2007; Mendelsohn 2003; Naranjo, 2005; OECD 2007; Romeis 2006; Torres and Ruberson, 2005; Torres and Ruberson, 2007; Yu 2011, Whitehouse 2005). Specifically , a meta - anal ys is 6 of the data collected from 42 field studies indicated that non - target invertebrates are generall y more abundant in Bt cotton and Bt corn fields than in non - transgenic fields managed with insecticides (Marvier 2007). In addition, a comprehensive review of short and long term field studies on the effects of inv ertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop cultivation (Sanvido 2007). Environmental exposure of non - target aquatic organisms is generall y dependent u

pon the input of crop debris into streams and waterway s. Because of the short - term exposure and rapid degradation of Cry proteins, there is limited opportunity for exposure 2 weeks after a Cry protein is introduced into the environment by crop debris or po llen shed (Jensen 2010 ; Wolt 2010). Therefore, the level of Cry proteins found in any surrounding aquatic ecosystem is expected to be below that at which biological activity

would be observed (Jensen 2010, Sy ngenta 2010). Assessment of insecticidal transgenic crops should include laboratory tests with indicator species to determine potential toxicity at doses of the toxin higher than would be anticipated under field conditions (Rose 2007). Syngenta submitted data from laboratory and field studies on non - target representative species at exposure levels in excess of that anticipated in the environment (Sy ngenta 2010, pp. 134 - 152). Data was submitted for two abo ve - ground arthropods (insidious flower bug ( Orius insidious )) and spotted ladybird beetle ( Coleomegilla maculata )), two soil dwelling arthropods (rove beetle ( Aleochara bilineata ) and ground or carabid beetle ( Poecilus cupreus )), a soil dwelling annelid (earthworm (

Eisenia fetida )), a pollinator (honeybee ( Apis mellifera )), two birds (Bobwhite quail ( Colinus virginianus ) and chicken ( Gallus gallus domesticus )), a mammal (mouse (

M us musculus )), an aquatic arthropod ( fresh water shrimp ( Gammarus fasciatus )), and a fish (catfish ( Ictalurus punctatus )). The data submitted in the petitio n indicate that no significant adverse effects were observed at the maximum test dose for any of the tested species.

5 Broad spectrum insecticides are ch emical insecticides which kill insects that are causing injury to plants and also kill other insects that are not causing injury to the plant. Insects that are inadvertently killed by the application of insecticide are called “non - target” insects. 6 M eta - analysis com

bines the results of several studies that address a set of related research hypotheses. The general aim of a meta - analy sis is to more powerfully estimate the true "effect size" as opposed to a smaller "effect size" derived in a single study unde r a given single set of assumptions and conditions.8

Event 5307 is expected to be similar with respect to the low potential harm to the environment (Sy

ngenta 2010). Based on the above information, APHI S has concluded that adverse impacts to non - target organisms exposed to Event 5307 corn are unlikely. **Potential for Transfer of Genetic Information with which Event 5307 Cannot**  **Interbreed** Horizontal gene transfer and expression of DNA from a plant species to bacteria is unlikely to occur (Keese 2008). First, many bacteria (or parts thereof) that are closel y associated w ith plants have been sequenced, including Agrobacterium and Rhizobium (Kaneko 2000; Kaneko 2002; Wood 2001). There is no evidence that these organisms contain genes derived from plants. Second, in cases where review of sequence data implied that horizontal gene tran sfer occurred, these events are inferred to occur on an evolutionary

time scale on the order o f millions of years (Brown 2003; Koonin 2001). Third, FDA has evaluated horizontal gene transfer from the use of antibiotic resistance 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 unlikel y to occur and thus poses no plant pest risk. **Potential for Event 5307 to have Altered Disease and Pest Susceptibilities**  APHI S assess ed whether Event 5307 is likely to have significantly altered disease and pest susceptibility . This assessment encompassed a thorough consideration of the introduced trait and disease and pest susceptibility data from Event 5307 field trials. Sy ngenta collected field trial data on Event 5307 from 2005 through 2009 (Sy ngenta 2011, Appendix A). Except for differences in rootworm damage, no observatio ns of differences were observed in pest damage or in disease susceptibility (Sy ngenta 2011, Table III - 1, Figure s III - 3, VIII - 1, VIII - 2, p. 101) . In f ield trials conducted in 2007 where gray leaf spot and northern corn leaf blight was present, Event 5307 did not show increased susceptibility to gray leaf spot or northern corn leaf blight diseases (Sy ngenta 2011 , Table VII - 8) . In 2009, no significant differences were observed in the disease ratings for eyespot, southern corn leaf blight and gray leaf spot (Sy

ngenta 2011, Table VII - 11). The Agrobacterium transformed plants used in the generation of Event 5307 were treated with an antibiotic to kill the Agrobacterium cells (Sy ngenta 2011, p. 26). Furthermore, DNA sequences derived from plant pests that were incorporated in Event 5307 do not result in the production of infectious agents or disease s ymptoms in plants, and so it is unlikely that Event 5307 could pose a plant pest risk. The description of the genetic modifications, including genetic elements, expression of the gene product and th eir functions for Event 5307 has been summarized above . APHI S considered whether corn constituents that differed between Event 5307 corn and other corn varieties might be sufficient to lead to increased susceptibility to pathogens or9

pests. The data presented in the petition indicate no difference in compositional and nutritional quality

of Event 5307 compared to conventional corn, apart from the presence of the eCry3.1Ab and PMIproteins (Sy ngenta 2011). None of the values for seed and forage composition c haracteristics were outside the range of natural variability of conventional corn seed and forage (Sy ngenta 2011, Table s VII - 13, VII - 14, VII - 15, VII 16, VII - 17, VII - 18, VII - 19, VII - 20, VI I - 21). Therefore, the composition of Event 5307 is not biologicall y different than conventional cor n (with the exception of the eCry3.1Ab and PMIproteins). Based on the known functions and mechanism of actions of these proteins (summarized in Sy ngenta 2011), none of these proteins are expected to directl y alter susceptibil ity

to plant pests. Thus, Event 5307 is expected to be susceptible to the same pests as conventional corn except for a reduction in rootworm damage. Corn is not a plant pest in the U.S., and the introduced DNA in Event 5307 is unlikely to pose a plant pes t risk. Based on the anal ys is of genetic modifications and their functions and field testing data submitted by the petitioner, APHI S concludes that there are no significant differences between Event 5307 corn and the non - transgenic counterparts relative to pest and disease susceptibility . **Conclusion** APHI S has prepared this plant pest risk assessment in order to determine if Event 5307 is unlikely to pose a plant pest risk. Based on the information provided by the applicant and the lack of plant pest risk from the inserted genetic material, weedy characteristics, aty pical responses to disease or plant pests in the field, effects on non - target s or beneficial organisms in the agro - ecos ys tem, and horizontal gene transfer, APHI S has concluded that Event 5307 is unlikely to pose a plant pest risk. **References** Baker, H.G. (1965) Characteristics and Modes of Origin of Weeds. In : The Genetics of Colonizing Species. H.G. Baker and G. L. Stebbins (eds.). pp. 147 - 172. Academic Press, New York and London. Brown, J.R. (2003) Ancient horizontal gene transfer. Nature Reviews Genetics 4:121 - 132. Calvi n , D . (2003) Western and Northern corn rootworm management in Pennsylvania . Pennsly vania State University

Fact Sheet. <http://ento.psu.edu/extension/factsheets/corn - rootworm> Accessed July 19, 20 11 . Cattaneo, M.G., Yafuso, C., Schmidt, C., Huang, C. - Y., Rahman, M., Olson, C., Ellers Kirk, C., Orr, B.J., Marsh, S.E., Antilla, L ., Dutilleul, P., Carrière, Y. (2006) Farm scale evaluation of the impacts of transgenic. Proceedings of the National Academy of Sciences of the United States of America 10:7571

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Kaneko, T. et al. (2000) Complete genome structure of the nitrogen - fixing sy mbiotic bacterium Mesorhizobium loti . DNA Research 7:331 - 338. Kaneko, T. et al. (2002) Complete genomic sequence of nitrogen - fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Research 9:189 - 197. Keeler, K. (1989) Can genetically engineered crops become weeds? Bio/Technology 7:1134 - 1139 . 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) Ho rizontal gene transfer in prokaryotes: quantification and classification . Annual Review of Microbiology 55:709 - 742. Marvier, M., McCreedy, C., Regetz, J. Kareiva, P. (2007) A meta - anal ys is of effects of Bt cotton and maize on nontarget invertebrates. Science 316: 1475

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