The Bt-maize Event 176 (tradenames NaturGard™ KnockOut™) was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. These novel plants produce a truncated version of the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subp. kurstaki strain HD-1. Event 176 is also genetically modified to express the bar gene cloned from the soil bacterium Streptomyces hygroscopicus, which encodes a phosphinothricin-N-acetyltransferase (PAT) enzyme. The PAT enzyme is useful as a selectable marker enabling identification of transformed plant cells as well as a source of resistance to the herbicide phosphinothricin (also known as glufosinate ammonium, the active ingredient in the herbicides Basta, Rely, Finale, and Liberty ). PAT catalyses the acetylation of phosphinothricin, and thus detoxifies phosphinothricin, eliminating its herbicidal activity. Delta-endotoxins, such as the Cry1Ab protein expressed in Event 176, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

Event 176 was produced by biolistic transformation of the inbred line CG00526 with two plasmids. One plasmid contained two copies of a 3’ truncated cry1Ab gene, each regulated by different promoter sequences. The cry1Ab open reading frame, corresponding to the sequence encoding the N-terminal 648 amino acids of the native Cry1Ab protein, was modified for optimal expression in plant cells. Green tissue expression of one copy of the cry1Ab gene was regulated by the phosphoenolpyruvate carboxylase promoter while expression of the other cry1Ab gene was controlled by a pollen specific promoter isolated from maize. Both genes employed 3’-polyadenylation sequences from the 35S transcript of cauliflower mosaic virus (CaMV). This plasmid also contained a copy of the beta-lactamase encoding bla gene under control of a bacterial promoter. The bla gene was not expressed in plant cells, but was employed as a selectable trait for screening bacterial colonies for the presence of the plasmid vector. The second plasmid contained a copy of the bar gene from the soil bacterium Streptomyces hygroscopicus which encodes the phosphinothricin N-acetyl transferase (PAT) enzyme. This enzyme is used as a selectable marker and confers resistance to glufosinate ammonium herbicide. Constitutive expression of the bar gene was under the control of the CaMV 35S promoter. Apart from the sequences encoding Cry1Ab and PAT, no other plant translatable DNA sequences were introduced into the plant genome.

The Introduced DNA
The cry1Ab gene, bar gene (used as a selectable marker and for herbicide tolerance) and a selectable marker for ampicillin resistance, beta-lactamase (bla) gene, were introduced into the maize chromosomes of Event 176.

The synthetic cry1Ab gene has approximately 65% homology at the nucleotide level with the native gene. The truncated Cry1Ab protein contains the insecticidal region of the native Cry1Ab as demonstrated by western blot analyses where similar bands were displayed at approximately 65 kDa for Event 176-expressed Cry1Ab and the native protein. Three additional immunoreactive proteins weighing approximately 60, 40 and 36 kDa were detected in the leaves of Event 176, but not in the pollen. It was suggested that these may represent breakdown products resulting from intrinsic proteolysis within the leaf tissue. Southern blot analysis indicated that there may be two or more copies of each plasmid integrated into the genome of Event 176. Southern blot analysis also confirmed the presence of the bar gene in all plant tissues.

Genetic Stability of the Introduced Trait
Segregation and stability data demonstrated that the cry1Ab and bar genes were tightly linked and stably inherited into the genome of Event 176 maize. The production of Cry1Ab and PAT proteins, in leaves and pollen of greenhouse-grown plants was determined to be stable over four successive backcross generations. Segregation analyses indicated that the resistance to ECB and herbicide tolerance traits co-segregate as linked Mendelian traits. A study of 3240 plants indicated that only five plants (0.15%) were identified as being tolerant to glufosinate ammonium but susceptible to damage by ECB larvae.

Expressed Material
Event 176 was transformed with two synthetic cry1Ab genes; one gene is linked to a specific promoter which confers expression in green tissue, while the other is linked to a pollen-specific promoter, resulting in expression in pollen. Expression of the Cry1Ab protein in green tissues was intended to render the plant resistant to first generation ECB larvae feeding on leaves while expression in pollen was intended to target second generation ECB larvae, which are known to feed on pollen. The glufosinate ammonium tolerance gene (bar gene) is linked to a constitutive promoter active in all plant tissues except pollen.

Cry1Ab production was quantified in leaves, pollen, roots and kernels among three genotypes. Expression in leaves, across genotypes, ranged from 0.596 to 1.159 µg/g (fresh weight) in seedlings, 0.530 to 3.029 µg/g (f.w.) at anthesis, 0.442 to 0.471 µg/g (f.w.) at physiological maturity, and 0.066 to 0.225 µg/g (f.w.) at senescence. Maximum expression in leaves was detected at either the vegetative or anthesis stages, depending on the genotype tested, and expression in all genotypes declined as plants senesced. Expression in pollen, across genotypes, ranged from 1.137 to 2.348 µg/g (f.w.). Expression of Cry1Ab in roots ( 0.008 µg/g f.w.), pith (<0.008 µg/g f.w.), and kernels (<0.005 µg/g f.w) was below levels of quantification. Whole plant expression at anthesis, as a percentage of total protein ranged from 0.00025 to 0.0014 % (f.w.).

While Southern blot analysis confirmed the presence of the bar gene in all plant tissues, expressed PAT protein was undetectable in leaves, pollen, roots or kernels of transgenic maize at a detection threshold of 0.2 ppm. The hydroxamic acid, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-2(4H)-one (DIMBOA) is the only putative endogenous toxin from maize and postulated to play a protective role against specific fungal and bacterial pathogens as well as insect pests. The levels of DIMBOA are normally highest in the leaf tissue of young plants and absent in kernels. The measured concentrations of DIMBOA in leaf tissue from transgenic 176 maize and untransformed control plants grown under identical conditions were statistically identical.

An ampicillin resistance gene (bla gene), regulated by a bacterial promoter, also present in the vectors used to transform Event 176, was not expressed in either leaf tissue or pollen as confirmed by assays and Northern blot analysis. Other regulatory DNA elements inserted to enhance expression levels of cry1Ab did not code for any other protein.

Field Testing
Field testing of Event 176 demonstrated that there were no significant differences observed between hybrids derived using original elite lines and Event 176 for the agronomic traits of yield, moisture at harvest, root lodging rating, ear height, plant height, heat units to silking or pollen shed. Other than resistance to ECB and tolerance to glufosinate ammonium herbicide, the disease, pest and other agronomic characteristics of Event 176 maize were comparable to non-transgenic lines of maize did not demonstrate any altered plant pest potential.

Outcrossing
Since pollen production and viability were unchanged by the genetic modification resulting in Event 176, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between Event 176 and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In North America, where there are few plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Cultivated maize, 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 of the genus Tripsacum.

Wild diploid and tetraploid members of Zea collectively referred to as teosinte are normally confined to Mexico, Guatemala, and Nicaragua; however, a fairly rare, sparsely dispersed feral population of teosinte has been reported in Florida. All teosinte members can be crossed with cultivated maize to produce fertile F1 hybrids. Nonetheless, in the wild, introgressive hybridization from maize to teosinte is currently limited, in part, by several factors including distribution, genetic incompatibility, differences in flowering time, developmental morphology, dissemination, and dormancy. First-generation hybrids are generally less fit for survival and dissemination in the wild, and show substantially reduced reproductive capacity which acts as a significant constraint on introgression.

In Central America, many of these species occur where maize might be cultivated, however, gene introgression from Event 176 under natural conditions is highly unlikely. It is further unlikely that potential introgression of European corn borer resistance or glufosinate tolerance traits from Event 176 would cause teosinte to become more weedy in the absence of glufosinate herbicide selection.

The genus Tripsacum contains up to 16 recognized species, most of which are native to Mexico, Central and South America, but three of which exist as wild and/or cultivated species in the U.S. 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 are able to withstand even the mildest winters. Furthermore, none of the sexually compatible relatives of maize in the U.S. are considered to be weeds in the U.S. therefore it is unlikely that introgression of the bar gene would provide a selective advantage to these populations as they would not be routinely subject to herbicide treatments.

Weediness Potential
No competitive advantage was conferred to Event 176, other than that conferred by resistance to European corn borer and herbicide tolerance to glufosinate. Maize is not a weed, and resistance to European corn borer or herbicide tolerance will not render maize weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified. Potential introgression from maize Event 176 into wild relatives is not likely to increase the weediness potential of any resulting progeny nor adversely effect genetic diversity of related plants any more than would introgression from traditional maize hybrids.

Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. Maize volunteers are not uncommon but are easily controlled by mechanical or by using herbicides that are not based on glufosinate as appropriate. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects
The history of use and literature suggest that the bacterial Bt protein is not toxic to humans, other vertebrates, and beneficial insects. This protein is active only against specific lepidopteran insects and no lepidopteran species are listed as threatened or endangered species in North America.

Studies were conducted on several nontarget organisms to determine the potential toxic effects of Cry1Ab protein on test organisms including adult honeybees (Apis melifera,), a predator lady beetle (Coleomegilla maculata), juveniles of the soil-dwelling invertebrate Collembola (springtails,Folsomia candida), earthworms (Eisenia foetida), juveniles of the freshwater invertebrate Daphnia magna, fall armyworm and black cutworm, Northern bobwhite chicks, and mice. Additional studies assessed the impact of Cry1Ab protein on the relative abundance of beneficial arthropods.

Results demonstrated that the larval development of honeybees and lady beetles were not affected when reared on pollen collected from Event 176 maize plants as compared to pollen from nontransgenic plants. Similarly, there was no effect on survival, immobilization, or sublethal toxicity reported for the small aquatic insect, Daphnia magna when exposed to pollen collected from Event 176. Survival rates, signs of toxicity, or loss of weight were not observed in earthworms exposed to leaf tissue from Event 176 maize as compared to the control treatments. Two lepidopteran insects (fall armyworm and black cutworm) that are not susceptible to native Cry1Ab were likewise not affected when feed Cry1Ab derived from leaf tissues. Three insects (ECB, corn earworm, and cabbage looper) that are susceptible to native Cry1Ab were also susceptible to plant-produced Cry1Ab. Results from high dose feeding studies of bobwhite quail fed a protein extract enriched in Cry1Ab isolated from Event 176 maize demonstrated no adverse effects on the bird.

Negative effects were observed in Collembola fed the Event 176 leaf protein (5 % mortality at 0.088 mg Cry1Ab /kg soil) while Collembolans fed with non-Bt maize protein were not adversely affected. The level of Cry1Ab was approximately 10 times greater than the maximum soil concentration that would occur if Event 176 plants were to be incorporated into the soil at anthesis. Under normal conditions, maize would be plowed under in the fall when plants have senesced, and the Cry1Ab protein in Event 176 would be present at very low levels at this time.

Acute oral toxicity studies were conducted with northern bobwhite quail and mice. These animals were fed Event 176-produced protein, and in the case of mice, were also fed bacterial-expressed protein. No mortality was observed and was not expected since Event 176-produced protein was shown to degrade very rapidly under simulated mammalian gastric conditions.

Small scale field studies demonstrated that the number of insects and insect diversity observed on plots planted to either Event 176 maize or a non-transformed counterpart were not significantly different. However, when compared to insect populations on plants treated with a common chemical insecticide (permethrin) versus Event 176 maize plants, the total numbers of beneficial insects (especially lady beetles) associated with Event 176 maize plants were higher.

Results from a study using an enriched leaf extract from Event 176 maize on the soil arthropod Collembola, Folsomia candida, showed that there was a reduction in adult survival and the number of offspring when fed concentrations of Cry1Ab protein at concentrations 200-fold higher than normal. This insect is one of a number of organisms that recycle plant debris in the field. The result was not totally unexpected since a related B. thuringiensis subsp. galleriae has been reported to kill Collembolla. Nonetheless, postharvest monitoring of field test with Event 176 maize plants showed no increase in visible amount of maize debris when compared to non-transgenic plants concluding that there should be no significant adverse effect on Collembola and no increase in maize plant debris as result of the cultivation of Event 176 maize.

In summary, it was concluded that cultivation of Event 176 maize should not have a significant potential to harm nontarget and beneficial organisms common to agricultural ecosystems, nor should it significantly impact species recognized as threatened or endangered.

Impact on Biodiversity
Event 176 had no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in North America is remote, it was determined that the risk of transferring genetic traits from Event 176 to species in unmanaged environments was insignificant.

Event 176 maize plants are not likely to eliminate the use of chemical insecticides which are traditionally applied to about 25 to 35% of the total maize acreage planted, since the primary target for most of these applications has been the coleopteran, corn rootworm. Event 176 maize may positively impact current agricultural practices used for insect control by 1) offering an alternative method for control of European corn borer (and potentially other Cry1Ab-susceptible pests of maize); 2) reducing the use of insecticides to control European corn borer and the resulting potential adverse effects of such insecticides on beneficial insects, farm worker safety, and ground water contamination; and 3) offering a new tool for managing insects that have become resistant to other insecticides currently used or expressed in maize, including other Bt-based insecticides.

Event 176 maize, along with glufosinate ammonium herbicides, is expected to positively impact current agricultural practices used for weed control by 1) offering growers a broad spectrum, post-emergent weed control system; 2) providing the opportunity to continue to move away from pre-emergent and residually active herbicides; 3) providing a new herbicidal mode of action in maize that allows for improved management of weeds which have developed resistance to herbicides with different modes of action; and 4) decreasing cultivation needs and increasing the amount of no-till acres. Volunteers of Event 176 can be easily controlled by selective mechanical or manual weed removal or by the use of herbicides with active ingredients other than glufosinate ammonium.

Other Considerations
In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in Canada and U.S. have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including Event 176, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

Dietary Exposure
The genetic modification of Event 176 maize will not result in any change in the consumption pattern for this product. Consequently, dietary exposure to this product is anticipated to be the same as for other lines of commercially available maize.

Nutritional Data
Results from proximate analysis, and analyses of fatty acid profiles, carotenoids, and amino acid profiles showed that Event 176 did not differ significantly from non-transformed isogenic lines, and indicated that there were no unintended effects on metabolic pathways. Comparisons of protein, fat, fibre and ash concentration of corn grain and whole plant material from Event 176 and the non-transgenic counterpart showed occasional significant differences in fat and protein content and whole plant ash content. The observed variation described was judged to be normal variation rather than due to the inserted novel trait.

Toxicity
Direct toxicity studies conducted using Cry1Ab and PAT test material did not reveal any deleterious effects. The amino acid sequence of the truncated Cry1Ab protein expressed in 176 maize is closely related the sequence of the same proteins that are present in strains of B. thuringiensis that have been used for over 40 years as commercial organic microbial insecticides. An analysis of the amino acid sequences of the inserted Cry1Ab protein and the PAT enzyme did not show homologies with known mammalian protein toxins and they are not judged to have any potential for human toxicity.

Allergenicity
The truncated Cry1Ab protein and the PAT enzyme expressed in 176 corn do not possess characteristics typical of known protein allergens. There were no regions of homology when the sequences of these introduced proteins were compared to the amino acid sequences of known protein allergens. Unlike known protein allergens, both of these proteins were rapidly degraded by acid and/or enzymatic hydrolysis when exposed to simulated gastric fluids. The Cry1Ab and PAT proteins are extremely unlikely to be
allergenic.

Diane E. Stanley-Horn , Galen P. Dively, Richard L. Hellmich, Heather R. Mattila, Mark K. Sears, Robyn Rose , Laura C. H. Jesse, John E. Losey, John J. Obrycki, and Les Lewis. (2001). Proc. Natl. Acad. Sci. USA Early Edition.

Draft Risk Analysis Report: Food derived from insect-protected Bt-176 corn

Decision Document DD96-09: Determination of Environmental Safety of Event 176 Bt Corn (Zea mays L.) Developed by Ciba Seeds and Mycogen Corporation

A. R. Zangerl, D. McKenna, C. L. Wraight, M. Carroll, P. Ficarello, R. Warner, and M. R. Berenbaum (2001). Proc. Natl. Acad. Sci. Early Edition

Opinion on the invocation by Germany of Article 16 of Council 90/220/EEC regarding the genetically modified BT-MAIZE LINE CG 00256-176 notified by CIBA-GEIGY (now NOVARTIS), notification C/F/94/11-03 (SCP/GMO/276Final - 9 November 2000) (Opinion adopted by written procedure following the SCP meeting of 22 September 2000)

COMMISSION DECISION of 25 April 2007 on the withdrawal from the market of Bt176 (SYN-EV176-9) maize and its derived products

Mark K. Sears, Richard L. Hellmich, Diane E. Stanley-Horn, Karen S. Oberhauser, John M. Pleasants, Heather R. Mattila, Blair D. Siegfried, and Galen P. Dively (2001). Proc. Natl. Acad. Sci. USA Early Edition

Outline of the biological diversity risk assessment report: Type 1 use approval for SYN-EV176-9

Preliminary report on the ecological impact of Bt corn pollen on the Monarch butterfly in Ontario.

NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGY INSECT RESISTANT CORN, 176

97/98/EC: Commission Decision of 23 January 1997 concerning the placing on the market of genetically modified maize (Zea mays L.) with the combined modification for insecticidal properties conferred by the Bt-endotoxin gene and increased tolerance to the herbicide glufosinate ammonium pursuant to Council Directive 90/220/EEC (Text with EEA relevance)

Petition for determination of nonregulated status of Ciba Seeds' corn genetically engineered to express the Cry1A(b) protein from Bacillus thuringiensis subspecies kurstaki

Biopesticide Fact Sheet: Bacillus thuringiensis Cry1Ab Delta-Endotoxin and the Genetic Material Necessary for Its Production (Plasmid Vector pCIB4431) in Corn [Event 176]

Memorandum to file concerning insect-resistant maize event 176.

USDA/APHIS Petition 94-319-01 for Determination of Nonregulated Status for Event 176 Corn

Bertheau, Y., Helbling, J.C., Fortabat, M.N., Makhzami, S., Sotinel, I., Audeon, C., Nignol, A.C., Kobilinsky, A., Petit, L., Fach, P., Brunschwig, P., Duhem, K. and Martin, P. (2009). Persistence of plant DNA sequences in the blood of dairy cows fed with genetically modified (Bt176) and conventional corn silage. J. Agric. Food Chem. 57: 509-516. [View abstract]

Betz, F.S., Hammond, B.G. & Fuchs, R.L. (2000). Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regulatory Toxicology 32, 156-173.

Li, Y., Meissle, M. and Romeis, J. (2008). Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green Lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae). PLoS ONE 3(8): e2909. [View abstract]  

Oliveira, A.P., Pampulha, M.E. and Bennett, J.P. (2008). A two-year field study with transgenic Bacillus thuringiensis maize: effects on soil microorganisms. Sci. Total Environ. 405: 351-357. [View abstract]

Brake, J. & Vlachos, D. (1998). Evaluation of transgenic event 176 “Bt” corn in broiler chickens. Poultry Science 77: 648-653. [View abstract]  

Faust, M. (1997). Study finds no Bt in milk. Integrated Crop Management – Special Livestock Edition, Fall, 1997. p6-7.