## Plant Pest RiskAssessment for MR162 Corn
Syngenta Biotechnology, Inc., has petitioned APHIS (APHIS number 07-253-01p) for a determination that genetically engineered (GE) corn (Zea mays) eventMIR162 is unlikely to pose a plantpest risk and, therefore, is no longer a regulated article under regulations at 7 CFRpart 340. This plantpestrisk assessment was conducted to determine whether MIR162 corn is unlikely to pose a plantpestrisk. IfAPHIS determines that MIR162 corn is not a plantpest, APHIS then has no regulatory authority
overthat organism under its regulations at 7 CFR part 340. 

### History ofDevelopment ofMIR162 Lepidopteran-Resistant Corn

Corn is susceptible to attack by a variety ofinsects from thetime it is planted until it is consumed as food orfeed (Table 1on page 12 in petition). Syngenta has developed MIR162 containing an insecticidal protein Vip3Aa20 (Vip = Vegetative insecticidal protein) that is resistantto the feeding damage caused by corn earworm (Helicoverpa zea), fall armyworm (SpodopteraJrugiperda), blackcutworm (Agrotis ipsilon), and western bean cutworm (Striacosta albicosta) larvae. Vip3Aa is produced by the bacteriumBacillus thuringiensis (Bt) (Estruch et al. 1996). Bacteria use Vip protein toxinsto kill insect prey (Estruch et al. 1996; Schnepfet al. 1998) which then serves as a nutritional source (de Maagd etal. 2001). Vip3Aa proteins are similarto certain Cry proteins (HOfte and Whiteley 1989) and are demonstrated to have toxic effects only on certain insects (Table 2.1 on p. 23 in Carozzi and Koziel 1997).
The mechanism by which Vipproteins exerttheir insecticidal activity has been studied and found to be similar, but not identical, to that which has been previously described for the BtCry proteins that are contained in several commercial insecticide formulations and plants engineered for insect resistance. The Vip and Cry proteins bind to different receptors in the insect (Lee et al. 2003), and the insecticidal activity ofVip3Aaproteins is limited to species within selected families ofthe orderLepidoptera (Table 27 on pages 74-75 in petition). For example, the Vip3Aa protein in MIR162 does not provide corn plantsprotection against damage caused by European corn borer (Ostrinia nubilalis) and
corn root worm (Diabrotica sp), the most widespread and damaging insect pests ofmaize in the U.S. Corn Belt. According to Syngenta, when MIR162 corn hybrids containing Vip3Aa are combined with European corn borerprotected corn, such hybrids have the potential toprovide growers the means ofprotectingtheir corn crops from damage caused by a broader range oflepidopteran pests (pages 74-75 in petition).

MIR 162 corn has been field tested under APHIS regulations since 1999. Data were provided in the petition for field trials completed prior to the petition submission.
Tolerance exemptions and conditional pesticide registrations have been granted for the plant-incorporatedprotectant in MIR162 corn and the genetic material necessary for its production. On August 6, 2008, the Environmental Protection Agency (EPA) granted an exemption from the requirement ofa tolerance for residues ofVip3Aaproteins (includingthe Vip3Aa20 variant) in oron food and feed commodities ofcom (73 FR45620-45624). Likewise, on April 30, 2009, EPA also approved the conditional registrations of Vip3Aa20 produced in MIR 162 comfor use as lepidopteran insecticide (74 FR 19956­ 19957). An exemption from the requirement oftolerance has been established for the selectable marker gene phosphomannose isomerase (PMI) protein in all crops (69 FR 26770-26775). Syngenta's food safety summary submittedto FDA indicated that food and feed derived from comevent MIR162 are as safe and nutritious as food and feed derived from conventional com. Atthe conclusion oftheirconsultation with FDA on December 9, 2008, the FDA concluded that it had "no further questions concerning grain and forage derived from comeventMIR162" (FDA BNF No. 000113). 

### Description ofInserted Genetic Material

MIR162 maize was produced by transformation ofimmature maize embryos using an Agrobacterium tumefaciensplant-pathogenic bacterium vector system that is disarmed of DNA sequences within the T-DNA (!ransfer-DNA), which upon integration into the plant genome ofinfected cells are normally responsible for the formation ofcrown gall tumors in plants. The disarmedAgrobacterium tumefaciens harbors a plasmid vector pNOV1300 that contained within its T-DNA vip3Aa19 and manA gene expression cassettes.
The first expression cassette consists offour genetic elements:



### • Z. mays polyubiquitin promoter and first intron (ZmUbiInt). Thispromoter provides constitutive expression in monocots (Christensen et al. 1992).

• Full length Vip3Aa19 gene. This protein coding region is a variant ofthe native vip3Aal gene (Estruch etal. 1996) from B. thuringiensis strain AB88. The vip3Aa19 gene was codon optimized for expression in maize (Murray etal. 1989). The vip3A19 encodes a Vip3Aa19 protein that has insecticidal activity against many lepidopteran insectpests.

• Intron #9 from the phosphoenolpyruvate carboxylase gene (iPEPC9) from Z. mays (Hudspeth and Grula 1989).

• 35S RNA Terminator sequence from the cauliflowermosaic virus genome. This sequence contains signals for termination oftranscription and directs polyadenylation (Francket at. 1980).
The second expression cassette consists ofthree genetic elements:

• ZmUbiInt (same as above).

• A manA gene from E. coli strain K-12. This gene encodes the enzyme phosphomannose isomerase (PMI) that catalyzes the interconversion ofmannose

### 26-phosphate to fructose-6-phosphate (Negrotto etal. 2000), which was used as a selectable marker duringtransformant selection.

• NOS (Nopaline Synthase) gene terminator sequence fromA. tumefaciens. This terminates gene expression by a polyadenylation site (Depicker et aL 1982).

The production ofPMI enzyme in the transformed tissue allows corntissue containing the vip3Aa19 and manA gene expression cassettesto be selected on medium containing the sugar mannose. The manA gene expression, whichproducesphosphoisomerase enzyme, confers no otherbenefitto the regenerated transformed corn plant. Syngenta provided evidence demonstrating thatthe final product does not contain any ofthe backbone sequences outside ofthe T-DNA borders from the transformation vector, pNOV1300.

Southern blot analyses and nucleotide sequencing demonstrated that MIR162 corn contains a single intact T-DNA insert in the corn genome. Furthermore, Southern blot analyses also demonstrated thatthe T -DNA insert contains: i) single copies ofa vip3Aa gene and a manA gene; ii) two copies ofthe ZmUbiIntpromoter; iii) one copy ofthe NOS terminator; and iv) no backbone sequences from transformation plasmid pNOV1300. Nucleotide sequencing additionally determined thatthe MIR162 maize TDNA insert did not locate within anyknownZ. mays gene. Further, no novel open reading frames were createdthat spanned eitherthe 5' or 3'junctionsbetweenthe T-DNA andZ. mays genomic sequences. Syngenta's characterization ofthe T-DNA in MIR 162 and Mendelian inheritance of transgene segregation dataprovide evidence for the functional stability and intactness of the twotransgene coding sequences over several breeding generations during the development ofMIR162 cornhybrids (pages 23- 46 in petition). However, the vip3A19 coding sequence analysis also revealedtwo single nucleotide changes in the coding sequence contained in the MIR162 maize T-DNA, as compared with the sequence present in the transformation plasmid pNOV1300. The new gene variant incorporated into theMIR162 maize genome has beendesignated as vip3Aa20. One ofthe mutations resulted in a single codon change forthe amino acid originally encoded, while the other mutation was a silent mutation (i.e., the amino acid produced did notchange). Mutational changes in genetic elements are very common and are the ultimate source of genetic variation found in nature, andthe single functional mutational change in the vip3Aa19 gene thatresulted in vip3Aa20 is within the range ofmutation rates observed in nature for plants (Kovalchuk et at. 2000). The single amino acid substitution between vip3Aa19 andvip3Aa20 occurs at position 129 (see the petitionpages47-48 for a detailed description). Based on Syngenta's laboratory bioassay results, the amino acid differences between vip3Aa19 and vip3Aa20 variants do not impact insecticidal activity against target insectpests, asthe position 129 occurs outside ofthe core protein domain involved in insecticidal activity (Estruch and Yu 2001; Lee etaL 2003). These dataalso indicate that no novel proteins, otherthan Vip3Aa20 and PMI, will be produced in MIR162 maize. These genetic characterization data demonstratethat, apart from the wellcharacterized change thatresulted in a single altered amino acid in the vip3Aa19 coding

3sequence, there are no unintended changes in the MIR162 corn genome as a result ofthe T-DNA insertion (see pages 23-46 ofpetition).


### Plant Pest RiskAssessment

MIR162 maize was producedbytransformation ofcorn tissue usingA. tumefaciens to introduce a gene that confers tolerance to certain lepidopteran (caterpillar) pests ofcom. BecauseA. tumefaciens is a plant pest and some ofthe regulatory sequences used to facilitate expression ofthese genes in com were derived from plant pests, the engineered
corn has been considered a regulated article under APHIS regulations at 7 CFRpart 340. APHIS administersthese regulations under the authority ofthe PlantProtection Actof2000 (PPA) (7 U.S.C. Sec 7701 etseq.). APHIS' authority to regulate genetically engineered organisms under the PPA is limited to those GEorganisms that are plantpests as defined under Section 14 ofthe PPA. APHIS regulations under 7 CFRpart 340.1 defines a plant pest as "Any living stage (including active and dormantforms) ofinsects, mites, nematodes, slugs, snails, protozoa, orotherinvertebrate animals, bacteria, fungi, other parasiticplants orreproductive parts thereof; viruses; orany organisms similarto or allied with any ofthe foregoing; orany infectious agents orsubstances, which can directly orindirectly injure orcause disease ordamage in orto any plants orparts thereof, orany processed, manufactured, orotherproducts ofplants."
Potential impacts to be addressed in this risk assessment are those thatpertain to the use ofMIR162 com and its progeny in the absence ofconfinement. Ofthe information requested by APHIS for submission ofapetition for nonregulated status (§ 340.6(c)(4», APHIS examined information submittedby the applicant related toplantpest risk characteristics, disease andpest susceptibilities, expression ofthe gene product, new enzymes or changes to plant metabolism, weediness ofthe regulated article, and any impacts on the weediness ofany otherplantwith which itcan interbreed. Furthermore, APHIS examined the effects ofthe regulated article on nontarget organisms, as MIR162
com is genetically engineered to produce a lepidopteran-specific toxin. Issues related to agricultural orcultivationpractices are in the Environmental Assessment for MIR162
com. 

### Potential Impacts ofGenetic Modifications on Altered Disease and Pest Susceptibilities

USDA-APHIS assessed whether MIR162 com is likely to have significantly increased disease and pest susceptibility. This assessment encompasses a thorough consideration of introducedtraits and interactions with pests and disease.
Com (Zea mays ssp. mays) is not a plant pest in the United States (USDA-APHIS 2000). Furthermore, none ofthe sequences derived from the plantpests (Agrobacterium and CaMV) thatwere incorporated into MIR 162 comresult in the production ofinfectious agents ordisease symptoms in plants, and so they are unlikely to pose a plant pest risk. The description ofthe genetic modifications, including genetic elements, expression of the gene product and their functions for MIR162 comhas been summarized above.

4Syngentaroutinely monitors their corn field trials for the fungal diseases gray leafspot, northern corn leafblight, and southern corn leafblight. The corn insects monitored include corn rootworm, corn flea beetle, grasshopper, stink bug, and othercoleopteran beetles (personal communication to Subray Hegde, APHIS, BRS 8/27/09). The data submitted by Syngenta indicated no meaningful differences between MIRI62 corn and the non-transgenic counterparts for disease, such as grey leafspot disease (petition Tables 22 and 23, pp. 65-66, and Supplementto Petition, Tables S-5 and S-6, pp. 7-8), and nontargeted insectpests (field test reports submitted to APHIS/BRS on notifications and permits, Table A.I,pages 127-128 in petition).
The data presented in the petition indicate no difference in compositional and nutritional quality ofMIR162 corn compared to conventional corn, apart from the presence of Vip3Aa20 and PMI proteins. Although some ofthe variables measured by the applicant showed statistically significantdifferences between MIR162 corn and the non-transgenic hybrid controls (Table 26 onpage 72 in petition), none ofthe values for the forage and grain composition characteristics were outside the range ofnatural variability of conventional corn reported by the International Life Sciences Institute Crop Composition Database (Ridley etal. 2004; ILSI 2006) or in the OECD consensus document on corn composition (OECD 2003). Therefore, the composition ofMIRI62 corn is not biologically different than conventional corn (with the exception ofthe Vip3Aa20 and PMI proteins). Based on the known functions and mechanisms ofactions ofthese proteins (summarized in the petition), neither ofthese proteins are expectedto directly alter susceptibility to plant pathogens. Thus MIR 162 corn is expected to be susceptible to the same plant pathogens as conventional corn.
The Vip3Aa20 protein will decrease the susceptibility ofMIR 162 corn to certain targeted insectpests (as noted below underPotentialImpacts on Target andNontarget Organisms), which could indirectly affectpopulations ofother insectpests on corn, and likewise plantpathogens that infect corn as a result offeeding damage. As noted in the petition (pg. 13) insects pests ofcorn play an important role in the transmission and dissemination ofpathogenic organisms during maize development. According to the Petitioner, "Ear, kernel, and cobrots occur wherevermaize is grown and canresult in reducedtestweight, poor grain quality, and mycotoxin contamination offood and feed. Fusarium kernel or earrot is the mostwidespread disease ofmaize ears and is frequently associated with insect feeding damage." They indicate that although crop losses attributable to 

### 0. nubilalis and Diabrotica infestations have been well characterized and
are significant, there is not as much quantitative information available onthe economic impacts ofothermajor insectpests ofmaize, specifically the leafand ear-feeding insects H. zea, S.frugiperda,A. ipsiion, and S. albicosta, which are the primary targetpests of Vip3A120 in MIR 162 corn. Thesepests are not as widespread as some corn pests, but crop infestations by these pests have the potential to significantly lower grain yield and quality. Data in the petition (petition Table 22 and 23, pp. 65-66) showed no significant difference in grain yield between a MIR 162 corn hybrid and a near isogenic control line in two years offield trials in a variety ofUS maize growing locations (6-10 locations per year) (petition Table 21, pg. 63). These data indirectly support that MIR 162 corn does not have increased susceptibility to insects orpathogens that directly or indirectly affect



### 5yield. In a variety offield studies, other insectprotected corn expressing Btproteins have been shown to have significantly lower levels ofcommon mycotoxin that are produced by fungal pathogens (Wu 2006); however, no data were provided on mycotoxin levels in MIR 162 corn. 

### Potential Impacts from Outcrossing (Gene Flow) to Sexually-compatible Wild Relatives

Gene flow is a natural biological process with significant evolutionary importance. A numberofangiosperm taxa are believed to be derived from hybridization orintrogression between closely related taxa (Grant 1981; Soltis and Soltis 1993;Rieseberg 1997; Hegde et al. 2006), and even in the existing floras, the occurrence ofhybridization or introgression is reportedto be widespread (Knobloch 1972; Stace 1987; Rieseberg and Wendel 1993;Peterson et al. 2002). Ithas been a common practice by plant breeders to artificially introgress traits from wild relativesinto crop plants to develop new cultivars. However, gene flow from crops to wild relatives is also thought ofas having a potential to enhance the weediness of wild relatives, as observed in rice, sorghum, sunflowerand few othercrops (see Table 1in Ellstrand etal. 1999).
APHIS evaluated the potential for gene introgression to occurfrom MIRl62 cornto sexually compatible wild relatives and considered whether such introgression would result in increased weediness. Cultivated corn, ormaize, Zea mays L. subsp. mays, is sexually compatible with othermembers ofthe genusZea, and to a much lesser degree with members ofthe genus Tripsacum (OECD 2003). Wild diploid and tetraploid members ofZea, collectively referred to as teosinte, are normally confined to the tropical and subtropical regions ofMexico, Guatemala, and Nicaragua. In the U.S. a fairly rare, sparsely dispersed feral population ofteosinte has been reported in Florida (USDA Plant database: http://plants.usda.gov/java/county?state

_name=Florida&statefips=12&symbol=ZEME).
The genus Tripsacum contains up to 16 recognized species, most ofwhich are native to Mexico, Central and South America, butthree (T. dactyloides, T.floridatum, and T. lanceolatum) exist as wild and/or cultivated species in the continental U.S (OECD 2003); and two taxa (T.fasciculatum and T. latifolium) also occur in Puerto Rico (PLANTS Database, accessed 7/1312009). Though many ofthese species occurwhere corn might be cultivated, gene introgression from MIR162 corn under natural conditions is highly unlikely. Hybrids ofTripsacum species with Zea are difficult to obtain outside ofa laboratory and are often sterile orhave greatly reduced fertility, and none ofthem can withstand even the mildest winters. Furthermore, none ofthe sexually compatible relatives ofcorn in the U.S. are consideredto be weeds in the U.S. (Holm et. al. 1979). Therefore, even in those instances ofaccidental gene flow between MIRI62 corn and wild relatives, the transgenes ofMIR162corn are unlikely to transform corn wild relatives into moreweedy species.

6Introgression ofgenes from corn into teosinte or Tripsacum species has not been described to occur in nature in the U.S. While some teosinte may be considered weeds in certain instances, they are also used by some farmers for breeding improved maize (Sanchez Gonzalez and Ruiz Corral 1997 and references therein). Teosinte is described
as being susceptible to many ofthe same pests and diseases that attack cultivated corn (Sanchez Gonzalez and Ruiz Corral 1997). In the wild, introgressive hybridization from
corn to teosinte is currently limited, in part, by several factors including geographic isolation, differing degrees ofgenetic incompatibility, differences in flowering time in
some cases, developmental morphology and timing ofthe reproductive structures, dissemination, and dormancy (Doebley 1990a and I990b; Galinat 1988; Ellstrand 2007). First·generation hybrids are generally less fit for survival and dissemination in thewild, and show substantially reduced reproductive capacity, which thus acts as a significant constraintto introgression. Data included in the petition demonstrated that there were no significantdifferences in viability and morphology ofpollen from collected from greenhouse-grown MIR162 hybrid plants and near-isogenic control plants (petition, Table 24 and Figure 20, pp. 67-68.); therefore, the outcrossing rate ofMIR 162 corn is not expected to be any different from other corn. Based on the data presented in the petition, MIR162 corn does not exhibit characteristics that cause it to be any weedierthan othercultivated corn (see below). Moreover, its potential impact due to the extremely limitedpotential for gene introgression intoteosinte is not expected to be any different than thatofother cultivated corn varieties.
Based on the above considerations, MIRI62 corn will not adversely impact sexually compatible wild relatives ortheirweediness characters.


### Potential Impacts Based on the Relative Weediness ofMIR162 Corn

In the U.S., corn is not listed as a weed (Crockett 1977; Holm et al. 1979; Muenscher 1980), nor is itpresentonthe Federal noxious weed list (7 CFRpart 360;**http://www.aphis.usda.gov/plant_health/plant~est_info/weeds/downloads/weedlist2006 .**  pdf). Furthermore, corn is grown throughoutthe world withoutany reportthat it is a serious weed orthat itforms persistentferal populations. Likemany domesticated crops, corn seed from a previous year's crop can overwinter and germinate the following year. For instance, the appearance ofcorn seedlings in soybean fields following a corn crop is a common occurrence. Manual orchemical measures are often applied to remove these volunteers, butthe plants that are notremoved do not typically result in feral populations in following years. Corn also possessesfew ofthe characteristics ofplantsthatare notably successful weeds (Baker 1965; Keeler 1989).

APHIS assessed whether MIRI62 corn is any more likely to become a weed than the isogenic nontransgenic corn line, or other corn varieties currently under cultivation. The assessment encompasses a thorough consideration ofthe basic biology ofcorn and an evaluation ofthe unique characteristics ofMIRI62 corn evaluated under field conditions. Syngentaconducted agronomic field trials during the 2005 and 2006 growing seasons across 6-10 locations representative ofthe major corn-growing areas ofthe upper midwestU.S. Forthe majority ofthe traits assessed, there were no statistically significant

7differences between MIR162-derived hybrids and their control counterparts. Results of the 2005 agronomic equivalence trials presented in Table 22 ofthe petition revealed only
one statistically significant difference between a control and MIR162 variant

- the average number ofgerminated plants per plot was slightly higher in the MIR162 plots compared to control plots. But the difference was only 3.2%, and is not considered to be ofbiological significance because the effect was not repeated in the 2006 trials, and there
was no effect ofgenotype observed in the seed germination and dormancy study (see Tables 18 and 19). No other differences in phenotypic characteristics that might contribute to enhanced weediness were observed between MIR162 and control lines for the wide range ofphenotypic endpoints assessed in these trials or in greenhouse or laboratory experiments (see petition Table 16, pp. 57-58, for the full list ofcharacteristics evaluated). These characteristics covered seed germination and dormancy, emergence, vegetative and reproductive growth, seed retention, and plant-ecological interactions. Furthermore, the genes inserted into MIR 162 comdo not confertolerance to herbicides; therefore, there is no change in the ability to control MIR 162 comas a weed on agricultural land as a result ofthe insertion ofthe foreign genes.
Based on the agronomic field data and literature survey about com weediness potential, MIR162 com lacks ability to persist as troublesome weed, and there would be no direct impact on current weed managementpractices for com cultivation. 

### Potential Impacts on Target and Nontarget Organisms, Including Beneficial Organisms

The mechanism by which Vip proteins exert their insecticidal activity has been studied and found to be similar, but not identical, to that which has been previously described for the Cry proteins. Formany decades microbial products containing Bt (the organism that produces the Cry1A protein) have been used to control insectpests on a commercial scale and for home garden applications (Glare 2000; Shelton 2002). Plants that were genetically engineered to express the Cry1A protein have ahistory ofsafe use in the U.S. Since the mid-1990s, com and cotton lines have been commercialized without substantiated reports ofsignificant deleterious impacts on non-target organisms (EPA 2008; OECD 2007). Vip3Aahas activity against several ofthe major lepidopteran pests ofcom, specifically: A. ipsilon, H. zea, 

### S. albicosta, and S.frugiperda (Table 27 on pages 74-75 in petition). Syngenta summarized data from field efficacy trials comparing the activity of MIR162 with that ofBtl 1com, hybrids ofBt 11 x MIR162, and conventional insecticide (Warrior®Insecticide) treated corn, relative to untreated controls, with regard to feeding damage from these insects (Petition Figure 21 on page 76 in petition). MIR162 alone has
no activity against 

### 0. nubilalisbut is efficacious in limiting feeding damage caused by the other four insectpests (mean damage ratings were at most 30%ofthat ofthe untreated controls in Petition Figure 21 on page 76 in petition). Whereas Bt11 com lines (with Cry proteins) are highly efficacious against 

### 0. nubilalis, they have limited or no activity against the other four insects. According to Syngenta, the combined-trait

8Bt1lxMIRI62 hybrids are very efficacious against all five insects (mean damage ratings
were at most about 10% ofuntreated controls (Figure 21 on pg. 76 in petition).
The Bt Toxin Nomenclature Committee currently lists 25 variants ofthe Vip3Aa protein. This narrow spectrum ofactivity for Vip3Aaproteins is a positive attribute from an ecological perspective, as maize hybrids containing a Vip3Aaprotein are unlikely to pose a risk to nontarget organisms inhabiting maize ecosystems. Like Cry proteins, Vip3Aa proteins are not expected to adversely affect non-target invertebrates, such as bees, and vertebrate organisms, including birds, mammals and humans, because they do not contain the receptor found in the midgut oftarget insects. Dataprovided in the petition (summarized in the Table 31, pg. 89) confirmed that in the bird, mammal, honey bee, above ground arthropod, and soil dwelling invertebrate studies, no observable adverse effects or differences in survival were noted at doses ofVip3A proteins that were well above those expected from exposure to the Vip3Aa20 protein from MIRI62 planted in the field. The nontarget above-ground arthropods and soil-dwelling invertebrates studied (lady bird beetles, green lacewings, minute pirate bugs, collembola, earthworms, and rove beetles) were considered to be representative ofthe com agro-ecosystem. Although not an endangered orthreatened species,Danausp/exippus (monarch butterfly) is a species ofhigh conservation interest, and there has been concern that it may be harmed by consuming pollen from transgenic insect-protected maize. The monarch is susceptible to CrylAb (Hellmich et al. 2001), the most common insecticidal protein in transgenic maize. However, the distribution ofthe monarch's food plant (Asclepias syriaca

- common milkweed), its pattern ofmigration, and the timing ofmaize anthesis
means that very few monarchs are exposed to harmful concentrations ofCry 1 Ab (Sears et al. 2001).
The exposure assessments used to assess the risks ofmaize containing Cry1 Ab to monarchs are also valid for MIRI62 maize. In addition, it has been shown that monarchs
are not susceptible to Vip3Aal. Lee et al. (2003) showed that trypsin protease digested Vip3Aai did not form pores in the midgut ofmonarchs; pore formation appears to be essential for toxicity and occurs in the guts ofinsects susceptible to Vip3Aal. These investigators also found no mortality ofmonarch butterfly in a surface diet bioassay limit test at 1000 ng/cm2

. MIR162 com, therefore, poses low risk to monarchs because of minimal hazard ofVip3Aa20 and low exposure to Vip3Aa20-containing pollen. Besides, the restriction oftoxicity ofVip3Aa20to Lepidoptera, and the minimal exposure of endangered Lepidopterato maize, indicates that Vip3Aa20 in MIRI62 maize is expected to have no harmful effects on any endangered or threatened species in the U.S. 

### Potential Impacts from Transferring Genetic Information from MIR162 Corn to Organisms with which It

### cannot Interbreed

APHIS examined the potential for the new genetic material inserted into MIRl62 com to be horizontally 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, including the creation ofmore virulent pathogens. The horizontal gene transfer between unrelated

9organisms is one ofthe most intensively studied fields in the bio-sciences since 1940, and the issue gained extra attention with the release oftransgenic plants into the environment (Droge et al. 1998). Potential risks from stable horizontal gene transfer (HGT) from genetically engineered organisms to another organism without reproduction orhuman intervention was recently reviewed (Keese 2008). Mechanisms ofHGT include conjugation, transformation and transduction, and other diverse mechanisms ofDNA and RNA uptake and recombination and rearrangement, most notably through viruses and mobile genetic elements. HGT has been a majorcontributorto the spread ofantibiotic resistance amongstpathogenic bacteria and the emergence ofincreased virulence in bacteria, eukaryotes and viruses and in the long run has contributed to majortransitions in evolution. 

### Potential for Horizontal Gene Transferto Bacteria or Fungi

The MIR 162 has two bacteria genes. Horizontal gene transfer and expression ofDNA from a plant species to other bacterial species is unlikely to occurbased onthe following observations. Although there are many opportunities for plants to directly interact with fungi and bacteria (e.g. as commensals, symbionts, parasites, pathogens, decomposers, or in the guts ofherbivores), there are almost no evolutionary examples ofHGTto bacteria from eukaryotes orfrom plants to fungi (as reviewed in 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 ofthe foreign DNA inserted into the nuclear genome ofMIR162 com is unlikely to occur. First, many genomes (or parts thereof) have been sequenced from bacteriathat are closely associated with plants including Agrobacterium and Rhizobium (Kaneko etal. 2000; Wood et al. 2001; Kaneko etal. 2002). There is no evidence thatthese organisms contain genes derived from plants. Second, in
cases where review ofsequence data implied that horizontal gene transfer occurred, these events are inferred to occur on an evolutionary time scale onthe order ofmillions ofyears (Koonin et al. 2001; Brown 2003). Third, transgene DNApromoters and coding sequences are optimized for plantexpression, notprokaryotic bacterial expression. Thus even if horizontal gene transferoccurred,proteins correspondingto the transgenes are not likely to beproduced. Fourth, the FDAhas evaluated horizontal gene transfer from the use of antibioticresistance marker genes and concluded thatthe likelihood oftransfer of antibiotic resistance genes from plantgenomes to microorganisms in the gastrointestinal tract ofhumans oranimals, or in the environment, is remote**http://vm.cfsan.fda.gov/'~dms/opa-ann2.html ).**  

### Potential for Horizontal Gene Transfer to Viruses

APHIS also considered whetherhorizontal transfer ofDNA from MIR162 comto plant viruses was likely to occur and would lead to the creation or selection ofa more virulent plant pathogen through recombination with other plant viruses. This issue has been considered before by other science review panels and government regulatory bodies (for a general review ofthe issue see Keese 2008). The only virus sequence contained within MIR 162 com is the 35S RNA terminator and this has not been implicated in viral recombination.
Therefore, APHIS concludes thathorizontal genetransfer is unlikely to occurand thus poses no significant environmental orplantpest risk. Finally, under natural conditions; no

10transfer ofan intact functional gene has been demonstrated to date (Miki and McHugh 2004). Therefore APHIS concludesthat horizontal gene transfer is unlikely to occur and thus poses no significant environmental orplantpest risk. 

### Conclusion

APHIS has reviewed and conducted a plant pest risk assessment on MIR162 corn. Due to the lack ofplantpest risk from the inserted genetic material, the lack ofweediness characteristics ofMIR162 corn, the lack ofatypical responses to disease orplant pests in the field, the lack ofdeleterious effects on non-targets orbeneficial organisms in the agro-ecosystem, and the lack ofhorizontal gene transfer, APHIS concludesthat MIR162
corn is unlikely to pose a plantpest risk.

11

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