Plant Pest Risk Assessment for MR162 Corn 

Syngenta Biotechnology, Inc., has petitioned APHIS (APHIS number 07-253-01p) for a 

determination that genetically engineered (GE) corn (Zea mays) event MIR162 is 

unlikely to pose a plant pest risk and, therefore, is no longer a regulated article under 

regulations at 7 CFR part 340. This plant pest risk assessment was conducted to 

determine whether MIR162 corn is unlikely to pose a plant pest risk. If APHIS 

determines that MIR162 corn is not a plant pest, APHIS then has no regulatory authority 

over that organism under its regulations at 7 CFR part 340. 

History of Development of MIR162 Lepidopteran-Resistant Corn 

Corn is susceptible to attack by a variety of insects from the time it is planted until it is 

consumed as food or feed (Table 1 on page 12 in petition). Syngenta has developed 

MIR162 containing an insecticidal protein Vip3Aa20 (Vip = Vegetative insecticidal 

protein) that is resistant to the feeding damage caused by corn earworm (Helicoverpa 

zea), fall armyworm (SpodopteraJrugiperda), black cutworm (Agrotis ipsilon), and 

western bean cutworm (Striacosta albicosta) larvae. Vip3Aa is produced by the 

bacterium Bacillus thuringiensis (Bt) (Estruch et al. 1996). Bacteria use Vip protein 

toxins to kill insect prey (Estruch et al. 1996; Schnepf et al. 1998) which then serves as a 

nutritional source (de Maagd et al. 2001). Vip3Aa proteins are similar to 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 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 Bt Cry 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 ofVip3Aa proteins is 

limited to species within selected families of the order Lepidoptera (Table 27 on pages 

74-75 in petition). For example, the Vip3Aa protein in MIR162 does not provide corn 

plants protection against damage caused by European corn borer (Ostrinia nubilalis) and 

corn root worm (Diabrotica sp), the most widespread and damaging insect pests of maize 

in the U.S. Corn Belt. According to Syngenta, when MIR162 corn hybrids containing 

Vip3Aa are combined with European corn borer protected corn, such hybrids have the 

potential to provide growers the means of protecting their corn crops from damage 

caused by a broader range of lepidopteran 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-incorporated protectant 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 of a tolerance for residues of Vip3Aa proteins (including 
the Vip3Aa20 variant) in or on food and feed commodities of com (73 FR 45620-45624). 

Likewise, on April 30, 2009, EPA also approved the conditional registrations of 

Vip3Aa20 produced in MIR 162 com for use as lepidopteran insecticide (74 FR 19956­

19957). An exemption from the requirement of tolerance has been established for the 

selectable marker gene phosphomannose isomerase (PMI) protein in all crops (69 FR 

26770-26775). Syngenta's food safety summary submitted to FDA indicated that food 

and feed derived from com event MIR162 are as safe and nutritious as food and feed 

derived from conventional com. At the conclusion of their consultation with FDA on 

December 9, 2008, the FDA concluded that it had "no further questions concerning grain 

and forage derived from com event MIR162" (FDA BNF No. 000113). 

Description of Inserted Genetic Material 

MIR 162 maize was produced by transformation of immature maize embryos using an 

Agrobacterium tumefaciens plant-pathogenic bacterium vector system that is disarmed of 

DNA sequences within the T-DNA (!ransfer-DNA), which upon integration into the plant 

genome of infected cells are normally responsible for the formation of crown gall tumors 

in plants. The disarmed Agrobacterium tumefaciens harbors a plasmid vector pNOV1300 

that contained within its T-DNA vip3Aa19 and manA gene expression cassettes. 

The first expression cassette consists of four genetic elements: 

• 	 Z. mays polyubiquitin promoter and first intron (ZmUbiInt). This promoter 

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 et al. 1996) from B. thuringiensis strain AB88. The 

vip3Aa19 gene was codon optimized for expression in maize (Murray et al. 1989). 

The vip3A19 encodes a Vip3Aa19 protein that has insecticidal activity against 

many lepidopteran insect pests. 

• 	 Intron #9 from the phosphoenolpyruvate carboxylase gene (iPEPC9) from Z. 

mays (Hudspeth and Grula 1989). 

• 	 35S RNA Terminator sequence from the cauliflower mosaic virus genome. This 

sequence contains signals for termination of transcription and directs 

polyadenylation (Franck et 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 of mannose2 
6-phosphate to fructose-6-phosphate (Negrotto et al. 2000), which was used as a 

selectable marker during transformant selection. 

• NOS (Nopaline Synthase) gene terminator sequence from A. tumefaciens. This 

terminates gene expression by a polyadenylation site (Depicker et aL 1982). 

The production of PMI enzyme in the transformed tissue allows corn tissue containing 

the vip3Aa19 and manA gene expression cassettes to be selected on medium containing 

the sugar mannose. The manA gene expression, which produces phosphoisomerase 

enzyme, confers no other benefit to the regenerated transformed corn plant. Syngenta 

provided evidence demonstrating that the 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 that the T -DNA insert contains: i) single copies of a vip3Aa 

gene and a manA gene; ii) two copies of the ZmUbiInt promoter; iii) one copy ofthe 

NOS terminator; and iv) no backbone sequences from transformation plasmid 

pNOV1300. Nucleotide sequencing additionally determined that the MIR162 maize TDNA insert did not locate within any known Z. mays gene. Further, no novel open 

reading frames were created that spanned either the 5' or 3' junctions between the T-DNA 

and Z. mays genomic sequences. 

Syngenta's characterization of the T-DNA in MIR 162 and Mendelian inheritance of 

transgene segregation data provide evidence for the functional stability and intactness of 

the two transgene coding sequences over several breeding generations during the 

development ofMIR162 corn hybrids (pages 23- 46 in petition). However, the vip3A19 

coding sequence analysis also revealed two 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 the MIR162 maize genome has been designated as vip3Aa20. One of the mutations 

resulted in a single codon change for the amino acid originally encoded, while the other 

mutation was a silent mutation (i.e., the amino acid produced did not change). 

Mutational changes in genetic elements are very common and are the ultimate source of 

genetic variation found in nature, and the single functional mutational change in the 

vip3Aa19 gene that resulted in vip3Aa20 is within the range of mutation rates observed in 

nature for plants (Kovalchuk et at. 2000). The single amino acid substitution between 

vip3Aa19 and vip3Aa20 occurs at position 129 (see the petition pages 47-48 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 insect pests, as the position 129 occurs outside of the core protein domain involved 

in insecticidal activity (Estruch and Yu 2001; Lee et aL 2003). These data also indicate 

that no novel proteins, other than Vip3Aa20 and PMI, will be produced in MIR162 

maize. These genetic characterization data demonstrate that, apart from the wellcharacterized change that resulted in a single altered amino acid in the vip3Aa19 coding 

3 
sequence, there are no unintended changes in the MIR 162 corn genome as a result ofthe 

T-DNA insertion (see pages 23-46 of petition). 

Plant Pest Risk Assessment 

MIR 162 maize was produced by transformation of corn tissue using A. tumefaciens to 

introduce a gene that confers tolerance to certain lepidopteran (caterpillar) pests of com. 

Because A. tumefaciens is a plant pest and some of the regulatory sequences used to 

facilitate expression of these genes in com were derived from plant pests, the engineered 

corn has been considered a regulated article under APHIS regulations at 7 CFR part 340. 

APHIS administers these regulations under the authority of the Plant Protection Act of2000 

(PPA) (7 U.S.C. Sec 7701 et seq.). APHIS' authority to regulate genetically engineered 

organisms under the PPA is limited to those GE organisms that are plant pests as defined 

under Section 14 of the PPA. APHIS regulations under 7 CFR part 340.1 defines a plant 

pest as "Any living stage (including active and dormant forms) of insects, mites, 

nematodes, slugs, snails, protozoa, or other invertebrate animals, bacteria, fungi, other 

parasitic plants or reproductive parts thereof; viruses; or any organisms similar to or 

allied with any of the foregoing; or any infectious agents or substances, which can 

directly or indirectly injure or cause disease or damage in or to any plants or parts 

thereof, or any processed, manufactured, or other products of plants." 

Potential impacts to be addressed in this risk assessment are those that pertain to the use 

of MIR 162 com and its progeny in the absence of confinement. Of the information 

requested by APHIS for submission of a petition for nonregulated status (§ 340.6(c)(4», 

APHIS examined information submitted by the applicant related to plant pest risk 

characteristics, disease and pest susceptibilities, expression of the gene product, new 

enzymes or changes to plant metabolism, weediness of the regulated article, and any 

impacts on the weediness of any other plant with which it can interbreed. Furthermore, 

APHIS examined the effects of the regulated article on nontarget organisms, as MIR162 

com is genetically engineered to produce a lepidopteran-specific toxin. Issues related to 

agricultural or cultivation practices are in the Environmental Assessment for MIR162 

com. 

Potential Impacts of Genetic 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 

introduced traits 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 of the sequences derived from the plant pests (Agrobacterium and 

CaMV) that were incorporated into MIR 162 com result in the production of infectious 

agents or disease symptoms in plants, and so they are unlikely to pose a plant pest risk. 

The description of the genetic modifications, including genetic elements, expression of 

the gene product and their functions for MIR 162 com has been summarized above. 

4 
Syngenta routinely monitors their corn field trials for the fungal diseases gray leaf spot, 

northern corn leaf blight, and southern corn leaf blight. The corn insects monitored 

include corn rootworm, corn flea beetle, grasshopper, stink bug, and other coleopteran 

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 leaf spot disease (petition Tables 

22 and 23, pp. 65-66, and Supplement to Petition, Tables S-5 and S-6, pp. 7-8), and nontargeted insect pests (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 

qual ity of MIR 162 corn compared to conventional corn, apart from the presence of 

Vip3Aa20 and PMI proteins. Although some of the variables measured by the applicant 

showed statistically significant differences between MIR162 corn and the non-transgenic 

hybrid controls (Table 26 on page 72 in petition), none of the values for the forage and 

grain composition characteristics were outside the range of natural variability of 

conventional corn reported by the International Life Sciences Institute Crop Composition 

Database (Ridley et al. 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 of the Vip3Aa20 and 

PMI proteins). Based on the known functions and mechanisms of actions of these 

proteins (summarized in the petition), neither of these proteins are expected to 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 of MIR 162 corn to certain 

targeted insect pests (as noted below under Potential Impacts on Target and Nontarget 

Organisms), which could indirectly affect populations of other insect pests on corn, and 

likewise plant pathogens that infect corn as a result of feeding damage. As noted in the 

petition (pg. 13) insects pests of corn play an important role in the transmission and 

dissemination of pathogenic organisms during maize development. According to the 

Petitioner, "Ear, kernel, and cob rots occur wherever maize is grown and can result in 

reduced test weight, poor grain quality, and mycotoxin contamination of food and feed. 

Fusarium kernel or ear rot is the most widespread disease of maize 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 on the economic 

impacts of other major insect pests of maize, specifically the leaf and ear-feeding insects 

H. zea, S. frugiperda, A. ipsiion, and S. albicosta, which are the primary target pests of 

Vip3A 120 in MIR 162 corn. These pests 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 of field trials in a variety of US 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 or pathogens that directly or indirectly affect 

5 
yield. In a variety of field studies, other insect protected corn expressing Bt proteins have 

been shown to have significantly lower levels of common 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 

number of angiosperm taxa are believed to be derived from hybridization or introgression 

between closely related taxa (Grant 1981; Soltis and Soltis 1993; Rieseberg 1997; Hegde 

et al. 2006), and even in the existing floras, the occurrence of hybridization or 

introgression is reported to be widespread (Knobloch 1972; Stace 1987; Rieseberg and 

Wendel 1993; Peterson et al. 2002). It has been a common practice by plant breeders to 

artificially introgress traits from wild relatives into crop plants to develop new cultivars. 

However, gene flow from crops to wild relatives is also thought of as having a potential 

to enhance the weediness of wild relatives, as observed in rice, sorghum, sunflower and 

few other crops (see Table 1 in Ellstrand et al. 1999). 

APHIS evaluated the potential for gene introgression to occur from MIRl62 corn to 

sexually compatible wild relatives and considered whether such introgression would 

result in increased weediness. Cultivated corn, or maize, Zea mays L. subsp. mays, is 

sexually compatible with other members of the genus Zea, and to a much lesser degree 

with members ofthe genus Tripsacum (OECD 2003). Wild diploid and tetraploid 

members of Zea, collectively referred to as teosinte, are normally confined to the tropical 

and subtropical regions of Mexico, Guatemala, and Nicaragua. In the U.S. a fairly rare, 

sparsely dispersed feral population of teosinte 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 of which are native to 

Mexico, Central and South America, but three (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 of these species occur where corn might be 

cultivated, gene introgression from MIR 162 corn under natural conditions is highly 

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 ofthem can 

withstand even the mildest winters. Furthermore, none of the sexually compatible 

relatives of corn in the U.S. are considered to be weeds in the U.S. (Holm et. al. 1979). 

Therefore, even in those instances of accidental gene flow between MIRI62 corn and 

wild relatives, the transgenes ofMIR162 corn are unlikely to transform corn wild 

relatives into more weedy species. 

6 
Introgression of genes 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 of the 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 of genetic incompatibility, differences in flowering time in 

some cases, developmental morphology and timing of the reproductive structures, 

dissemination, and dormancy (Doebley 1990a and I 990b; Galinat 1988; Ellstrand 2007). 

First·generation hybrids are generally less fit for survival and dissemination in the wild, 

and show substantially reduced reproductive capacity, which thus acts as a significant 

constraint to introgression. Data included in the petition demonstrated that there were no 

significant differences in viability and morphology of pollen 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 of MIR 162 corn is 

not expected to be any different from other corn. Based on the data presented in the 

petition, MIR 162 corn does not exhibit characteristics that cause it to be any weedier than 

other cultivated corn (see below). Moreover, its potential impact due to the extremely 

limited potential for gene introgression into teosinte is not expected to be any different 

than that of other cultivated corn varieties. 

Based on the above considerations, MIRI62 corn will not adversely impact sexually 

compatible wild relatives or their weediness characters. 

Potential Impacts Based on the Relative Weediness of MIR162 Corn 

In the U.S., corn is not listed as a weed (Crockett 1977; Holm et al. 1979; Muenscher 

1980), nor is it present on the Federal noxious weed list (7 CFR part 360; 

http://www.aphis.usda.gov/plant_health/plant~est_info/weeds/downloads/weedlist2006. 

pdf). Furthermore, corn is grown throughout the world without any report that it is a 

serious weed or that it forms persistent feral populations. Like many domesticated crops, 

corn seed from a previous year's crop can overwinter and germinate the following year. 

For instance, the appearance of corn seedlings in soybean 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 typically result in feral populations 

in following years. Corn also possesses few ofthe characteristics of plants that are 

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 of the basic biology of corn and an 

evaluation of the unique characteristics ofMIRI62 corn evaluated under field conditions. 

Syngenta conducted agronomic field trials during the 2005 and 2006 growing seasons 

across 6-10 locations representative of the major corn-growing areas of the upper midwest U.S. For the majority ofthe traits assessed, there were no statistically significant 

7 
differences between MIR162-derived hybrids and their control counterparts. Results of 

the 2005 agronomic equivalence trials presented in Table 22 of the petition revealed only 

one statistically significant difference between a control and MIR162 variant - the 

average number of germinated 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 

of biological significance because the effect was not repeated in the 2006 trials, and there 

was no effect of genotype 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 of phenotypic endpoints assessed in these trials or in greenhouse or 

laboratory experiments (see petition Table 16, pp. 57-58, for the full list of characteristics 

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 com do not confer tolerance to herbicides; 

therefore, there is no change in the ability to control MIR 162 com as a weed on 

agricultural land as a result of the insertion of the 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 management practices 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. For many decades microbial products containing Bt (the organism that 

produces the Cry1A protein) have been used to control insect pests on a commercial scale 

and for home garden applications (Glare 2000; Shelton 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, com and cotton lines have been commercialized without 

substantiated reports of significant deleterious impacts on non-target organisms (EPA 

2008; OECD 2007). 

Vip3Aa has activity against several of the major lepidopteran pests of com, 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 of Btl 1 com, 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. nubilalis but is efficacious in limiting feeding damage caused by 

the other four insect pests (mean damage ratings were at most 30% of that of the 

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 

8 
Bt1lxMIRI62 hybrids are very efficacious against all five insects (mean damage ratings 

were at most about 10% of untreated controls (Figure 21 on pg. 76 in petition). 

The Bt Toxin Nomenclature Committee currently lists 25 variants of the Vip3Aa protein. 

This narrow spectrum of activity for Vip3Aa proteins is a positive attribute from an 

ecological perspective, as maize hybrids containing a Vip3Aa protein 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 of target insects. Data provided 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 of Vip3A 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 of the com agro-ecosystem. 

Although not an endangered or threatened species, Danaus p/exippus (monarch butterfly) 

is a species of high 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 of the monarch's food plant (Asclepias 

syriaca - common milkweed), its pattern of migration, and the timing of maize anthesis 

means that very few monarchs are exposed to harmful concentrations of Cry 1 Ab (Sears 

et al. 2001). 

The exposure assessments used to assess the risks of maize containing Cry 1 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 of monarchs; pore formation appears to be 

essential for toxicity and occurs in the guts of insects susceptible to Vip3Aal. These 

investigators also found no mortality of monarch butterfly in a surface diet bioassay limit 

test at 1000 ng/cm2

. MIR 162 com, therefore, poses low risk to monarchs because of 

minimal hazard of Vip3Aa20 and low exposure to Vip3Aa20-containing pollen. Besides, 

the restriction of toxicity ofVip3Aa20 to Lepidoptera, and the minimal exposure of 

endangered Lepidoptera to 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 of more virulent pathogens. The horizontal gene transfer between unrelated 

9 
organisms is one of the most intensively studied fields in the bio-sciences since 1940, and 

the issue gained extra attention with the release of transgenic 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 or human 

intervention was recently reviewed (Keese 2008). Mechanisms ofHGT include conjugation, 

transformation and transduction, and other diverse mechanisms of DNA and RNA uptake and 

recombination and rearrangement, most notably through viruses and 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. 

Potential for Horizontal Gene Transfer to Bacteria or Fungi 

The MIR 162 has two bacteria genes. Horizontal gene transfer and expression of DNA 

from a plant species to other bacterial species is unlikely to occur based on the following 

observations. Although there are many opportunities for plants to directly interact with fungi 

and bacteria (e.g. as commensals, symbionts, parasites, pathogens, decomposers, or in the 

guts ofherbivores), there are almost no evolutionary examples ofHGT to bacteria from 

eukaryotes or from 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 bacteria that are closely associated with plants including 

Agrobacterium and Rhizobium (Kaneko et al. 2000; Wood et al. 2001; Kaneko et al. 2002). 

There is no evidence that these organisms contain genes derived from plants. Second, in 

cases where review of sequence data implied that horizontal gene transfer occurred, these 

events are inferred to occur on an evolutionary time scale on the order of millions ofyears 

(Koonin et al. 2001; Brown 2003). Third, transgene DNA promoters and coding sequences 

are optimized for plant expression, not prokaryotic bacterial expression. Thus even if 

horizontal gene transfer occurred, proteins corresponding to the transgenes are not likely 

to be produced. 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 resistance genes from plant genomes to microorganisms in the gastrointestinal 

tract of humans or animals, 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 whether horizontal transfer of DNA from MIR162 com 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 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 that horizontal gene transfer is unlikely to occur and thus 

poses no significant environmental or plant pest risk. Finally, under natural conditions; no 

10 
transfer of an intact functional gene has been demonstrated to date (Miki and McHugh 

2004). Therefore APHIS concludes that horizontal gene transfer is unlikely to occur and 

thus poses no significant environmental or plant pest risk. 

Conclusion 

APHIS has reviewed and conducted a plant pest risk assessment on MIR162 corn. Due to 

the lack of plant pest risk from the inserted genetic material, the lack of weediness 

characteristics of MIR162 corn, the lack of atypical responses to disease or plant pests in 

the field, the lack of deleterious effects on non-targets or beneficial organisms in the 

agro-ecosystem, and the lack of horizontal gene transfer, APHIS concludes that MIR162 

corn is unlikely to pose a plant pest risk. 

11 
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