Decision Document DD2006-59
Determination of the Safety of Dow AgroSciences Canada Inc.’s Insect Resistant and Glufosinate - Ammonium Tolerant Corn (Zea mays L) Event DAS-06275-8

A synthetic cry1F gene was developed to maximize it’s expression in corn, and introduced into the Hi-II hybrid. The gene codes for a truncated, core insecticidal protein with a very high degree of similarity to the B. thuringiensis var. aizawai native protein. The Cry1F protein expressed in event DAS-06275-8 is identical to the Cry1F protein expressed in corn line 1507, that is authorized for unconfined release and livestock feed in Canada (Decision Document DD2002-41). The protein expressed by B. thuringiensis var. aizawai is insecticidal to some lepidopteran species after cleavage in the insect’s gut to a bio-active, trypsin resistant core. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually results in cell lysis and insect death.

Samples of corn event DAS-06275-8 tissues were collected at various growth stages from six representative field trial sites in the US and Canada. The expression levels were measured at various stages of plant growth by enzyme-linked immunosorbent assay. Average Cry1F protein expression in nanograms protein per milligram dry weight tissue (ng/mg dwt) was as follows: 15 to 30.9 across growth stages in leaf, 6.75 to 8.82 across growth stages in root, 7.61 in stalk, 1.04 in grain, 4.58 in pollen and 13.9 in forage. As expected, the levels of Cry1F decreased in senescent corn tissues and the Cry1F protein was previously shown to degrade readily in soil.

The Cry1F protein expressed in corn event DAS-06275-8 is identical to the Cry1F protein expressed in corn line 1507. Studies submitted in the corn line 1507 application have shown that, unlike many allergens, the Cry1F protein degrades readily in simulated gastric fluid. The Cry1F protein is not biologically active following exposure to elevated temperature (> 75ºC). A search for amino-acid sequence similarity between the Cry1F protein and known allergens revealed no significant amino-acid homologies based on sequence identity of 8 or more contiguous amino acids.

Additionally, the Cry1F protein expressed in event DAS-06275-8 was demonstrated not to be glycosylated, providing additional evidence that Cry1F does not have the properties of known allergens.

Tests were done to demonstrate the equivalency of the Cry1F protein expressed in corn event DAS-06275-8 tissues with the bacterially-produced Cry1F protein used in the non-target organism tests and biochemical assays. The Cry1F protein extracted from corn tissues or from the bacteria was found to be biochemically equivalent. The Cry1F protein from both expression sources was sensitive to protease cleavage, resulting in a core truncated form of about 65 kDa. Both proteins were shown to be of similar immunological reactivity and lacked detectable glycosylation. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and N-terminal sequencing were used to determine the sequence equivalency of the two proteins.

The glufosinate-ammonium tolerance gene (bar gene) engineered into corn event DAS-06275-8 codes for the enzyme phosphinothricin N-acetyltransferase (BAR). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound.

The bar gene was originally isolated from Streptomyces hygroscopicus, an aerobic soil bacterium. The BAR enzyme is therefore naturally occurring in the soil. More generally, acetyltransferase enzymes are ubiquitous in nature.

The bar gene expressed in corn event DAS-06275-8 is linked to a constitutive promoter. Samples of corn event DAS-06275-8 tissues were collected at various growth stages from six representative field trial sites in the US and Canada. The expression levels were measured at various stages of plant growth by enzyme-linked immunosorbent assay. BAR protein expression in nanograms protein per milligram dry weight tissue (ng/mg dwt) was as follows: 129.2 to 224.2 across growth stages in leaf, 61.1 to 70.0 across growth stages in root, 103.3 in stalk, 5.94 in grain, 0.73 in pollen and 106.9 in forage. As expected, the BAR protein levels decreased in senescent corn tissues.

Protein allergens are normally resistant to digestion unlike the BAR protein which was shown to be rapidly digested under simulated gastric conditions.

The amino acid sequence of the BAR protein shows no significant homology to toxins in the Genbank database or allergens (based on sequence identity of 8 or more contiguous amino acids) from standard protein sequence databases.

The bar gene was expressed in a bacterial expression system and the resulting enzyme was used to perform toxicology studies and as a standard in the determination of protein expression from the modified plant. The BAR protein expressed in corn event DAS-06275-8 was compared to the bacterial-produced protein. The BAR protein extracted from corn tissues or from the bacteria was found to be biochemically equivalent. Both proteins were shown to be of similar molecular weight and immunological reactivity and lacked detectable glycosylation. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and N-terminal sequencing were used to determine the sequence equivalency of the two proteins.

The truncated cry1F gene expression cassette contains the complete Cry1F coding region with a truncated promoter region and intact terminator region. Sequencing of endogenous corn DNA 521 base pairs upstream and 2059 base pairs downstream of the truncated insert showed that an endogenous maize gene has not been interrupted by the insert. Upstream sequencing did not reveal any known promoter sequences. A study by Salgueiro et al. (2000) suggest that transcription of the cry1F gene could be directed by the truncated ubi intron present in the genetic construct.

The inheritance and stability of each introduced trait was determined using a combination of Southern blot analysis, lateral-flow immunoassays for expressed Cry1F protein and herbicide tolerance bioassay for expressed BAR protein.

The genetic stability of the insert within one stage in the breeding process, designated as the BC4S1 generation, and the linkage between the genotype and phenotypic trait expression was demonstrated. The stability of the insert across 2 additional generations was also confirmed. Segregation analyses across 4 additional generations were performed to determine the inheritance of the insect resistance and herbicide tolerance traits.

The results of the analysis are consistent with the finding of a single active site of insertion that segregates according to the Mendelian laws of genetics.

Dow AgroSciences Canada Inc. has provided the CFIA with a method for detection and identification of corn containing the 6275 transformation line.

CFIA evaluated data submitted by Dow AgroSciences Canada Inc. on the reproductive and survival biology of corn hybrids derived from event DAS-06275-8 and determined that vegetative vigour, time to maturity and seed production were within the normal range of expression of these traits currently displayed by commercial corn hybrids.

No competitive advantage was conferred to these plants, other than that conferred by resistance to the target pests and tolerance to glufosinate-ammonium herbicide. These traits were demonstrated not to render corn weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified.

The above considerations, together with the fact that the novel traits have no demonstrable effects on weediness or invasiveness, led the CFIA to conclude that corn event DAS-06275-8 has no altered weed or invasiveness potential compared to currently commercialized corn.

Some of the genetic elements introduced into corn event DAS-06275-8 were derived from Agrobacterium tumefaciens, but in all cases genes responsible for pathogenic qualities of the bacteria were not introduced. Therefore, the introduction of this genetic material would not be expected to result in corn event DAS-06275-8 expressing novel pathogenic characteristics.

Based on these points, the CFIA has determined that corn event DAS-06275-8 does not display any altered plant pest potential.

The Cry1F B.t. protein produced in corn event DAS-06275-8 is active only against specific lepidopteran insects; no lepidopteran species which are listed as threatened or endangered species in Canada will have significant exposure to the Cry1F protein produced by widespread cultivation of corn plants derived from event DAS-06275-8.

The Cry1F protein expressed in event DAS-06275-8 is identical to the Cry1F protein expressed in a previously authorized corn line, line 1507. Pollen and grain are likely sources of non-target exposure to Cry1F protein expressed in corn. There is a 3 to 6-fold reduction Cry1F expression in pollen compared to line 1507. Cry1F expression in grain of both lines is similar. Cry1F protein was previously shown to degrade readily in the soil.

Data from dietary toxicity studies on non-target organisms submitted in support of line 1507 application have shown that Cry1F protein has no effect on honeybees, green lacewing larvae, ladybird beetles, daphnia, collembola, parasitic hymenoptera, earthworm, bobwhite quail and mice, when ingested at doses greatly exceeding the levels of exposure to Cry1F protein from line 1507 tissues. It was also demonstrated that the Cry1F protein exhibits virtually no toxicity to larvae of the monarch butterfly at levels as high as 10 mg toxin/ml diet, a level which greatly exceeds anticipated environmental exposure, based on field trial data. Since pollen expression for line 6575 is substantially lower than for line 1507, there are extra margins of safety for event DAS-06275-8 over the previous line.

In addition, a new dietary toxicity study with the rainbow trout has been provided by Dow AgroSciences Canada Inc. No mortality or sublethal effects were observed over 8 days when the fish were fed daily with a standard fish diet containing 100 mg of Cry1F protein per kg of diet, supporting the lack of toxicity of event DAS-06275-8 to fish.

The herbicide tolerance trait of event DAS-06275-8 is conferred by expression of the phosphinothricin N-acetyltransferase (BAR). An acute oral gavage study was used to test the toxicity of BAR protein to mice. No mortality or abnormal clinical observations were noted over 2 weeks after the mice received a dose of 3250 mg of BAR protein per kg body weight. Phosphinothricin N-acetyltransferase enzymes, including the BAR protein expressed in event DAS-06275-8, have been studied extensively and have been found to be safe for food and feed uses.

Corn is not known for the production of significant levels of endogenous toxins and the transformation line that produced event DAS-06275-8 would not be expected to induce their synthesis. Corn is however, known to produce low levels of trypsin inhibitor and phytic acid. The levels of these compounds in event DAS-06275-8 were found to be equivalent to levels found in the control lines. The genetic modification, therefore did not alter the expression of endogenous toxins.

Based on the above, the CFIA has determined that the unconfined release of corn event DAS-06275-8, when compared with currently commercialized corn, will not result in altered impacts on non-target organisms.

Corn event DAS-06275-8 targets certain lepidopteran pest species, but it has been demonstrated to be safe for non-target organisms. The control of agricultural pest species is a common practice in Canada that is not restricted to the environmental release of PNTs, therefore the reduction in local pest species as a result of the release of corn hybrids containing event DAS-06275-8 does not present a significant change from existing agricultural practices.

The use of broad spectrum herbicides has the intended effect of reducing local weed populations within agricultural fields and this may reduce local weed species biodiversity, and possibly other trophic levels which utilize these weed species. It must be noted however that reduction in weed biodiversity in agricultural fields is not unique to the use of herbicide tolerant crops, and is a common practice in virtually all modern agricultural systems.

The CFIA has therefore concluded that the potential impact on biodiversity of corn event DAS-06275-8 does not present a significantly altered impact in comparison to corn varieties currently being grown in Canada.

The following IRM design is intended to reduce or delay European corn borer resistance to the Cry1F protein. A component of the IRM strategy that will be used with corn event DAS-06275-8, is the establishment of a refuge of ECB-susceptible corn within or near the Bt corn field. Should resistant insects occur, they would then be able to mate with susceptible insects to keep the frequency of resistance genes diluted in the insect population.

CFIA believes that sound management practices and IRM strategies can significantly reduce and delay the development of Cry1F resistant ECB populations, however the ECB populations must be monitored for the development of resistance in a regular and consistent manner. CFIA understands that Dow AgroSciences Canada Inc. has developed and will implement an insect resistance management plan that includes the following key components:

Notes: Corn event DAS-06275-8 also targets corn earworm, black cutworm and fall armyworm, however no modification is required to the present IRM design to address resistance in these pests since there are no significant over wintering populations of these insects in Canada.

The Plant Biosafety Office periodically audits compliance with the IRM requirements.

Composition of grain and whole plant from corn line TC6275 was compared with a control line with the same genetic background. Whole plant analysis included proximates, ADF, NDF, Ca and P, while grain analysis included proximates, major fatty acids, amino acids, vitamin A, folic acid, tocopherols, B vitamins, minerals, secondary metabolites (inositol, raffinose, furfural, P-coumaric acid, ferulic acid) and anti-nutrients (phytic acid and trypsin inhibitor). In grain, there was significantly lower P in line TC6275 than control, but all of the values were within literature values. Measurements for several of the analytes, although similar across treatments, were outside of the range of literature values. These included Ca, vitamin A, and total tocopherols.

Secondary Metabolites and Anti-Nutritional Factors

Concentration of inositol, raffinose, furfural, P-coumaric acid, ferulic acid, phytic acid and trypsin inhibitor was shown to be equivalent in control vs event DAS-06275-8 corn grain.

The applicant has demonstrated that the nutritional composition of corn event DAS-06275-8 is equivalent to control corn lines.

PAT is a highly substrate specific enzyme that has been well defined. Exposure to PAT protein is not new. The bar gene is isolated from Streptomyces hygroscopicus a common soil bacterium. The pat gene is present in the environment with no known adverse effects on humans and animals. In addition, PAT from the bar gene has been expressed in various crops authorized in Canada. The PAT protein from the bar gene also does not share any biologically relevant significant homology with known toxins or allergens. Studies indicate that the protein was heat and pH labile, and was inactivated within one minute when subjected to typical mammalian stomach and intestinal conditions. A mouse acute oral toxicity study using the bacterially expressed PAT protein indicated that there were no adverse effects at 3250 mg/kg body weight.

From the information provided by Dow AgroSciences Canada Inc., the Cry1F and the PAT proteins are unlikely to be novel toxins or allergens. Based on the predicted exposure levels and the results of the above tests, no significant risk to livestock and workers/by-standers is expected from exposure to the Cry1F and PAT proteins.

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