Please see the EU relevant links below.

Method for detection: Event specific real-time quantitative PCR based method for DAS-4Ø278-9 maize; the detection method is validated on the single-trait event using genomic DNA extracted from seeds of DAS-4Ø278-9 maize. Reference material: ERM®-BF433 accessible via the Joint Research Centre (JRC) of the European Commission. The relevant links are provided below.

• Inheritance studies conducted indicated that Mendelian segregation exists. • New expression proteins are expressed in low levels. • It is compositionally equivalent to its non-transgenic counterpart. • No evidence of similarity or homology was found with known toxic proteins. • There is no evidence of expression of known allergenic substances for the proteins expressed in the event. It is concluded that the event is substantially equivalent to its conventional counterpart, therefore, it is as safe and no less nutritious than conventional commercial varieties.

Dow AgroSciences Australia Limited has submitted an application to FSANZ to vary
Standard 1.5.2 – Food produced using Gene Technology – in the Australia New Zealand Food Standards Code (the Code) to include food from a new genetically modified (GM) corn line, DAS-40278-9. The corn has been modified to be tolerant to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase inhibitors (“fop” herbicides) such as quizalofop-P-ethyl.
Tolerance to these herbicides is conferred by expression in the plant of the aad-1 gene,
encoding aryloxyalkanoate dioxygenase (AAD-1), which inactivates herbicides having a
common aryloxyalkanoate structure The aad-1 gene is derived from Sphingobium
herbicidovorans, a common soil bacterium.
Corn is not a major crop in Australia or New Zealand. Domestic production is supplemented by the import of a small amount of corn-based products, largely as high-fructose corn syrup, which is not currently manufactured in either Australia or New Zealand. Such products are processed into breakfast cereals, baking products, extruded confectionery and food coatings. Other corn products such as cornstarch are also imported and used by the food industry for the manufacture of dessert mixes and sauces. Corn may also be imported in finished products such as corn chips and canned corn.
DAS-40278-9 corn will be grown in North America and is not intended for cultivation in
Australia or New Zealand. Therefore, if approved, food from this line may enter the
Australian and New Zealand food supply as imported food products.

The applicant requested from CTNBio technical advice for the commercial release of genetically modified maize DAS-40278-9 which confers tolerance to the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) and certain herbicides inhibitors of acetyl coenzyme A carboxylase (ACCase ) Aryloxyphenoxypropionate (AOPP) for the purpose of its release into the environment for the cultivation, production, handling, transfer, marketing, import, export, storage, consumption, release and disposal of the genetically modified organism and its derivatives for commercial purposes. CTNBio considers that: 1) The available information made it possible to properly evaluate the biosafety of genetically modified maize DAS-40278-9; 2) Scientific studies conducted to evaluate biosafety, agronomic and phenotypic characteristics, as part of the risk assessment of this GMO, included several ecosystems of representative regions for maize culture in Brazilian territory; 3) The phenotype of the transformed plants is equivalent to the original plant phenotype conventional in terms of human and animal health and safety for plants and the environment; (4) The commercial release of genetically modified maize DAS-40278-9 is not likely to cause significant degradation of the environment or human and animal health

molecular traditional methods

Please see decision document weblinks.

Based on the risk assessment, it can be concluded that the event shows the same risks as its conventional counterpart. Therefore the National Technical Biosafety Committee for GMO use exclusively in Health and human consumption (CTNSalud) recommends its authorization.

Indonesia National Agency of Drug and Food certified food safety for GM Maize event DAS-40278-9 (tolerance to 2 4-dichlorophenoxyacetic acid (2 4-D),and to aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitor

Competent National Authority: Ministry of Agriculture-Jehad, Agricultural Research, Education and Extension Organization (AREEO). Risk Assessment file is uploaded. https://bch.cbd.int/en/database/RA/BCH-RA-IR-115739/1

Please see the link below (in Japanese).

Please refer to the Risk Assessment Report

Authorization by COFEPRIS: 102

DAS-40278-9 corn produces aryloxyalkanoate dioxygenase-1 (AAD-1), an enzyme which confers resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and certain aryloxyphenoxypropionate (AOPP, or "fop") herbicides. The herbicide tolerance was achieved through transformation of a conventional corn variety.

UI OECD: DAS-4Ø278-9 During the risk assessment of this GMO based on existing knowledge to date, no toxic or allergic effects neither substantial nutritional changes are observed. The event is as safe as its conventional counterpart. For more detail please find attached the risk assessment summary in this page.

In conducting a safety assessment of food derived from herbicide-tolerant corn line DAS-40278-9, a number of criteria have been addressed including: a characterisation of the transferred gene, its origin, function and stability in the corn genome; the changes at the level of DNA, protein and in the whole food; compositional analyses; evaluation of intended and unintended changes; and the potential for the newly expressed protein to be either allergenic or toxic in humans. No potential public health and safety concerns have been identified in the assessment of insect-protected corn DAS-40278-9. On the basis of the data provided in the present application, and other available information, food derived from corn DAS-40278-9 is considered to be as safe for human consumption as food derived from conventional corn cultivars.

Simplified approval procedure: Through Ministry of Agriculture and Livestock Resolutions 1030 and 1071 there was stated a differentiated treatment for the commercial release of novel GE crops and for GE crops that have been approved in third countries, whose scientific, technical and safety characteristics are well-founded. Paraguayan Ministry of Agriculture’s Resolutions authorize taking into consideration the decision documents from third countries with regard to both human and animal food safety in the cases where these evaluations have been based on Codex Alimentarius, such as the Guidelines for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants and carried out in countries with time-tested regulatory systems and transparent procedures. Concerning environmental safety, assessments are accepted for GE crops that besides having been authorized for commercial planting in countries with sound regulatory systems, include in the decision documents considerations as follows: that the GE crop under review has been studied under different environmental conditions, behaving in the same way as the conventional non-GE counterpart; that it will be managed in an agronomic manner similar to any GE or conventional hybrid/variety of the species; another aspect is that Paraguay is not center of origin of that crop, and finally two relevant characteristics are that there are no related weeds in Paraguay with which the GE crop could cross-breed and that the main target pests and the main non-target arthropod species present in Paraguay have been taken into account in the GE risk assessment carried out in those countries. The Commercial Release Opinion of the National Commission for Agricultural and Forestry Biosafety (CONBIO), in its substantial part states: "...Recommends technically: (1) The commercial release of the event DAS-40278-9 (2) In case of detection of an unexpected effect, the company is obliged to inform CONBIO".

On January 20, 2017, Dow AgroSciences BV Philippine Branch submitted corn DAS 40278-9 for direct use as food and feed, or for processing, as original application under the DOST-DA-DENR-DOH-DILG Joint Department Circular (JDC) No. 1 Series of 2016.

 

After reviewing the Risk Assessment Report and attachments submitted by the applicant, the assessors namely: Scientific and Technical Review Panel (STRP), BPI Plant Products Safety Services Division (BPI-PPSSD) and Bureau of Animal Industry- Biotech Team (BAI-BT), concurred that corn DAS 40278-9 is as safe for human food and animal feed as its conventional counterpart.

STRP ASSESSMENT AND RECOMMENDATIONS Based on the documents submitted by the applicant: A. Host Organism The STRPs agree that Corn is a major item of grain grown for food and feed. Based on the combined compositional analysis from the literature, for human consumption , the grain contains (by dry weight basis) 63.3-89.8 % carbohydrates, 6.0- 17.3 % protein, 1.2-18.8 % total fat 8.3-35.3 % total dietary fiber and 0.62-6.28 % ash. For forage, it contains 66.9-94.5 % carbohydrates, 3.14-15.9 5 protein, 0.296-6.7 5 total fat. 19.0-62.8 % total dietary fiber and 1.3-10.5 % ash. Corn is also the source of some anti-nutrients like trypsin inhibitors, raffinose, phytic acid and secondary metabolites (coumaric acid, ferulic acid, furfural and inositol). The STRPs also agree that Corn in not known to be a toxicant, except for improperly stored grain that can be infested by insects and toxin-producing molds. Corn grain is not a source of allergen. However, the pollen of the corn plant is known be allergenic to hypersensitive individuals. According to Astwood et al, 1996. The potential pollen allergens known are Zea m1 and Clone C13. The pollen specific cDNAs of these are similar to those found in rye (Lol P1 sequence) and olives (ole e1) (Villalba et al, 1993). Pollen is shed at specific time points in the growing season. Since the subject of this application is corn grain, then pollen is not expected to be included in the importation. Further, the STRPs also noted that Corn has been used a primary source of food and consumed as food. In US, 90% of the total corn production is used as feed grain (meals, gluten feed, etc.). Estimates of both single serving (acute or short term intake) and repeat dose (chronic or average daily intake) corn exposures are available for consumption pattern that are relevant to the consumer. The intake of animal dietary burdens for livestock (beef, dairy, pig) and poultry are presented in tabulated form. Consumption pattern for livestock and poultry has been provided and described sufficiently. The introduction of the novel food (DAS 40278-9 Corn) is not expected to change as it was found to be substantially equivalent to conventional corn as a result of nutrient composition analyses. B. Donor Organism The STRPs concur that the inserted protein expressing aad-1 from a fragment of 6236 bp linear DNA was described. This was a fragment from the vector DNA plasmid pDAS1740. The regulatory sequences that included the promoter (Zm Ubi) the terminator (Zm Per 5 3’-UTR) were both from corn. The enhancers represented by the matrix attachment regions from tobacco (RB7 MAR v3 and v4 that flanked the aad-1 were adequately described in the patent documents. The promoter and terminator sequences were both expected to be recognized by the plant because they came from the same source and therefore ensure positive expression of the inserted gene. The MARS were included to increase consistency of expression and avoid any possible silencing (non-expression) of the inserted gene. The STRPs also agree that the aad-1 encodes Aryloxyalkanoate dioxygenase 1 which in the presence of α- ketoglutarate is known to degrade phenoxy auxins such as 2,4-D to dichlorophenol and eventually to succinate and carbon dioxide. These two products are common intermediated in cellular metabolism and are therefore not harmful by themselves. S. herbicidovorans could therefore use the phenoxy auxins and other xenobiotics as source of carbon for growth. Other substrates for this dioxygenase are the family of aryloxyphenoxypropionate (AOPP)–acetyl CoA inhibitors or “fop” pesticides. Further, they also agree that S. herbicidovorans, the source organism for the aad-1 gene is a gram-negative soil bacterium. Due to their biodegraduve and biosynthetic capabilities, the sphingomonads have been used for a wide range of biotechnological applications, including bioremediation of environmental contaminants and production of extracellular polymers such as sphingans which are used extensively in the food industry. There is no known pathogenicity and allergenicity of the encoded protein. C. Transformation System The STRPs all agree that the method of transformation was described and referenced as the Whisker-mediated direct DNA transfer using silicone carbide fiber directly penetrating the cell wall and directly incorporating the desired DNA. The target of genetic modification was the nuclear genome for stable and whole plant expression. The experimental procedure was adequately described and a schematic diagram was provided. A summary of the genetic elements are presented in tabulated form, indicating the locations on plasmid pDAS-1740 Tsp 1 fragment, size and description. he plasmid map of pDAS-1740 with all the genetic elements identified was presented. There was no carrier or helper DNA that was used in the introduction of the 8512 bp linear PTU from pDAS 1740 into the embryogenic cell suspension cultures to produce Event DAS 40278-9. E. Inserted DNA The molecular characterization of DAS-40278-9 corn was performed by Southern blot analyses. The result demonstrated that the transgene insert in DAS-40278-9 corn occurred as a single integration of a single intact copy of aad-1 expression cassette from plasmid pDAS 1740. The insert is stably integrated and inherited across and within the breeding generations. No plasmid backbone sequences are present in DAS-40278-9 corn. here was no indication of truncations in the Southern blots. The number and expected sizes of the specific hybridizing bands were consistently observed were consistently observed.There was also no indication of deletions in the Southern blots. F. Genetic Stability The STRPs concur that Southern blot analyses were conducted with five (5) distinct generations (T3, T4, BC3S3, and BC3S2) of DAS-40278-9 corn. Results across all DAS-40278-9 corn samples indicated stable inheritance of the intact single copy insert across multiple generations of DAS040278-9 corn. Segregation in T1, T2, BC1, BC2 BC3 and BC3S1 of DAS-40278- 9 corn were performed on leaf tissues through Southern blotting and immunoassay for the expressed AAD-1. The typical Mendelian inheritance ratio of 3:1 was observed. From the BC3S1 line for example, 65 tested positive for AAD-1 protein expression and 20 were null segregants. The aad-1 probe hybridized to each of the each 65 plants that tested positive for AAD-1. The 20 null segregants did not show this hybridization band, indicating the absence or non-inheritance of the inserted aad-1. G. Expressed Material A field expression study was conducted in 6 sites planted with DAS-40278-9 hybrid corn (BC3S1- A & E). Four treatments of the DAS-40278-9 corn expression study included 3 herbicide treatments as follows: AAD-1 Unsprayed, AAD-1 + Quizalofop, AAD-1 + 2,4-D, and AAD-1 + Quizalofop and 2,4-D. Results are presented showing the levels of AAD-1 protein (ng/mg tissue dry weight) measured in DAS-40278-9 corn indicating the range, mean and standard deviation. The average expression values ranged from 2.87 ng/mg dry weight in R1 stage root to 127 ng/mg in pollen tissue. For the plots sprayed and unsprayed with 2,4-D and quizalofop herbicides, no AAD-1 protein was detected in the control tissues across the 6 locations. A summary of the AAD-1 protein concentrations (average across sites) in various corn matrices are shown in tabulated form. The expression values were similar for the sprayed treatments as well as for the plots sprayed and unsprayed with 2,4-D and quizalofop herbicides. No AAD-1 protein has been detected in the control tissue across the 6 locations. H. Toxicological Assessment The STRPs agree that the digestibility of AAD-1 protein was tested in vitro using simulated gastric fluid (SGF). Samples were analyzed via SDS-PAGE and Western blot. The results demonstrated that AAD-1 protein was readily digested or inactivated (not detected at 30 seconds in SGF). Heat inactivation tests were also done, the lowest temperature used was 50oC for 30 min. These conditions already inactivated 97 % of the enzyme activity. The common cooking conditions applied to corn processing covers this and with the average field expression level of the AAD-1 present at 4.81 ng/mg tissue, processed products from DAS-40278-9 corn can be expected to be inactivated. Human consumption of raw corn is not the norm. The STRPs also agree that BLASTp Search summary of proteins in the alignmenmts with AAD-1 is presented and adequately described. None of the protein alignments returned by the BLASTp search are associated with toxicity. It was concluded that the AAD-1 proteins expressed in DAS-40278-9 corn contains no significant sequence similarity with any known toxic protein that is harmful to man and animals. Further, an acute oral toxicity study was conducted with AAD-1 protein in mice at a dose loevel of 2000 mg AAD-1/kg b.w. All the animals survived and gained weight. No adverse effects and clinical signs were observed by study termination on day 15. It was concluded that the acute oral LD50 of AAD-1 protein in mice was greater than 2000 mg/kg b.w., hence AAD-1 protein is not a health risk concern. The protein equivalency was also done by comparing aad-1 expression in transgenic corn with that of the Pseudomonas fluorescens using SDS-PAGE/Western blot/glycoprotein detection/MALDI-TOF MS/ and tandem mass spectrometry. The products of the analyteshave been shown to be biochemically equivalent. Biochemical equivalency was established by the procedures previously described. I. Allergenicity Assessment The STRPs agree that the digestibility of AAD-1 protein was tested in vitro using simulated gastric fluid (SGF). Samples were analyzed via SDS-PAGE and Western blot. The results demonstrated that AAD-1 protein was readily digested or inactivated (not detected at 30 seconds in SGF). Heat inactivation tests were also done, the lowest temperature used was 50oC for 30 min. These conditions already inactivated 97 % of the enzyme activity. The common cooking conditions applied to corn processing covers this and with the average field expression level of the AAD-1 present at 4.81 ng/mg tissue, processed products from DAS-40278-9 corn can be expected to be inactivated. Human consumption of raw corn is not the norm. The STRPs also agree that BLASTp Search summary of proteins in the alignmenmts with AAD-1 is presented and adequately described. None of the protein alignments returned by the BLASTp search are associated with toxicity. It was concluded that the AAD-1 proteins expressed in DAS-40278-9 corn contains no significant sequence similarity with any known toxic protein that is harmful to man and animals. The AAD-1 protein is not glycosylated as proven by glycoprotein staining. The AAD-1 has a molecular weight of 33 kDa as shown by electrophoretic separation. Further, an acute oral toxicity study was conducted with AAD-1 protein in mice at a dose loevel of 2000 mg AAD-1/kg b.w. All the animals survived and gained weight. No adverse effects and clinical signs were observed by study termination on day 15. It was concluded that the acute oral LD50 of AAD-1 protein in mice was greater than 2000 mg/kg b.w., hence AAD-1 protein is not a health risk concern. No serum screening was reported in the references possibly because of no evidence of similarity in sequence to known allergens. Please see above. J. Nutritional Data The STRPs agree that proximate analysis for grain show no differences across the six sites between control and transgenic lines were observed for fat ash, NDF and TDF. Fiber was within the reported literature range. A significant overall treatment effect was found for moisture but this did not show significance in the paired t-test or after adjustment for FDR. The differences in the values are not considered biologically meaningful because these were statistically low and in most cases was resolved after applying adjustment for false discovery rate (chance). Lastly, the values obtained still fall within those reported in the literature. Corn grain from Event DAS-40278-9 can therefore be considered substantially equivalent to conventionally-bred corn in terms of proximate. They also agree that there were also no significant differences in proximate analysis of forage, in control and transgenic lines for moisture, ADF, NDF, Ca and P. In comparison with the control line, protein content was lower in the transgenic line unsprayed and sprayed with quizalofop. On the other hand, ash content in the transgenic line sprayed with 2,4-D and quizalofop was highest. Carbohydrate was higher in the unsprayed and quizalofop-sprayed transgenic line. The differences are not of biological significance because there was no calculated treatment effect and the values still fall under the range of literature values. The value for carbohydrates was estimated by difference and has no significant FDR adjusted p-value and should therefore be of no biological concern. Furthermore, across –site analysis for levels of fatty acids in the grain gave values for 8:0 to 15:1 and 16:1 to 17:1 fatty acids that were below the limit of quantitation (LOQ). Values for 16:0 palmitic and 18:0, stearic acids where not significantly different and showed no overall treatment effect for the control and transgenic lines. Additionally the values were within the literature ranges. Anti-nutrients like raffinose was below the LOQ for the transgenic and non-transgenic control lines, as presented in table 24 of the summary. There was no significant difference found in the levels of trypsin inhibitor in the transgenic lines (4.87 to 5.45 % d.w.b) as compared to the non-transgenic line (5.08 %d.w.b). K. Recommendation Find scientific evidence that the regulated article applied for human food and animal feed use is as safe as its conventional counterpart and shall not pose any significant risk to human and animal health. BPI-PPSSD ASSESSMENT AND RECOMMENDATION Corn DAS 40278-9 was developed by Dow AgroSciences B.V., through the use of recombinant DNA technology. The said event was developed through Whisker’s – mediated direct DNA transformation of corn cells with pDAS1740 plasmid vector carrying, the aad-1 gene that encodes AAD-1 protein that provides herbicide tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides). Host Organism (Zea mays L.) Corn (Zea mays L.) has been widely consumed as staple food for humans and feed ingredient for animals. It is used in food products such as oil, grit, meal, flour, ethanol, syrup and starch as well as feeds such as hulls, gluten and hominy (OECD, 2002). Humans consume corn mostly in the form of corn-based ingredients such as high fructose corn syrup, starch, sweeteners, cereals, oil and alcohol. In terms of the feeds, it is commonly consumed in the form of corn silage (forage), gluten meal, gluten feed and distillers dried grains. In 2014, the daily per capita consumption index of corn in the Philippines is 60.08 grams/day, while the daily per capita calories supply is 213.88 grams (PSA, 2015). Corn is a source of key nutrients such as amino acids, fatty acids, carbohydrates, vitamins, minerals, and fiber (OECD, 2002). It is also known to contain anti-nutrients such as phytic acid, 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoaxin-3(4H)-one (DIMBOA), raffinose, trypsin and chymotrypsin inhibitors, and secondary plant metabolites such as furfural, ferulic acid and p-coumaric acid. These anti-nutrients and secondary metabolites have been historically present in corn at levels that would not cause the food to be unsafe. History of safe use was attributed to corn. It is known to produce no significant amount of toxins and anti-nutrients. It is not a common allergenic food; however, some reports had stated gastrointestinal and respiratory allergenic reactions. Transgenic Plant (DAS-40278-9 Corn) DAS-40278-9 corn has been reviewed and approved for food and/or feed use in many countries including Australia (Food, 2011), Brazil (Food and Feed, 2015), Canada (Food and Feed, 2012), Colombia (Food, 2014; Feed, 2013), European Union (Food and Feed, 2017), Japan (Food and Feed, 2012), Malaysia (Food and Feed, 2017), Mexico (2011), New Zealand (2011), South Africa (Food and Feed, 2012), South Korea (Food and Feed, 2014), Taiwan (2011), and United States of America (2011) (ISAAA). The event, DAS-40278-9 was developed to express AAD-1 proteins derived from Sphingobium herbicidovorans (Dow AgroSciences, 2014). The protein confers tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides). The transformation method is through Whisker’s – mediated direct DNA transfer with plasmid vector pDAS1740 into the corn line Hi-II. The plasmid vector contains the aad-1 gene expression cassette which contains two (2) matrix attachment region (MAR) obtained from Nicotiana tobacum (RB7 MAR), ubiquitin promoter isolated from Zea mays, synthetic, plant optimized version of an aryloxyalkanoate dioxygenase gene isolated from Sphingobium herbicidovorans, 3’ untranslated region from Zea mays peroxidase gene, and six (6) intervening sequences. Donor Organisms (Sphingobium herbicidovorans) Sphingobium herbicidovorans is a gram negative soil bacterium which has the ability to use phenoxy auxin and AOPP herbicides as carbon sources for growth (Dow AgroSciences, 2014). Sphingomonads are widely distributed in nature and was found in land, water, plant root systems, clinical specimens, etc. It has history of use in terms of bioremediation of environmental contaminants and production of extracellular polymers such as sphingans which are extensively used in food industry. The donor organisms of other genetic elements included in the plasmid vector pDAS1740 includes Nicotiana tobacum and Zea mays (Dow AgroSciences, 2014). History of safe use has been attributed to Z. mays since it is being widely consumed as staple food of several countries worldwide and is not a common allergenic food nor a source of toxicants. No food safety concern with regards to the other donor organisms used in the transformation since the regulatory sequences obtained from these organisms are not being expressed in DAS-40278-9. The only protein expressed in DAS-40278-9 is the aryloxyalkanoate dioxygenase-1 (AAD-1) protein encoded by aad-1 gene. AAD-1 has an alpha ketoglutarate-dependent dioxygenase activity (Dow AgroSciences, 2014). Inserted DNA Southern blot analyses using restriction enzymes such as EcoR I, Nco I, Sac I and Fse I/Hind III and aad-1, ZmUbi1 promoter and ZmPer5 terminator probes confirmed that the observed fragment sizes of each probe corresponds with the predicted fragment sizes of each probe of DAS-40278-9 genomic DNA and pDAS1740 (Dow AgroSciences, 2014). The results of analyses showed that DAS-40278-9 genome contains only a single insertion of the T-DNA from the plasmid pDAS1740. Also, no specific hybridization bands were detected in the negative control samples in any of the restriction enzyme and probe combinations. This indicates that the single insert in DAS-40278-9 corn contains an intact single copy of aad-1 gene. No truncations, deletions or rearrangements were identified. Southern blot analyses using Nco I and Sac I restriction enzymes on the backbone probes confirmed that no plasmid backbone sequences from pDAB1740 were integrated into DAS-40278-9. Genetic Stability The multigenerational stability of the introduced traits was assessed through Southern Blot Analysis of genetic samples from five generations (T3, T4, BC3S1, BC3S2 and BC3S3) of DAS-40278-9 (Dow AgroSciences, 2014). Results showed that aad-1 gene is stably inherited across multiple generations of DAS-40278-9. Segregation is assessed by Southern blot analysis and protein detection of individual plants from a BC3S1 line of DAS-40278-9. Chi-square analysis indicated that the segregation ratio of the plants with positive transgene insert versus negative transgene insert is consistent with the 3:1 segregation ratio characteristic of Mendelian inheritance pattern of a single dominant trait. Expressed Material (Cry34Ab1, Cry35Ab1 and PAT proteins) AAD-1 protein has specific mode of action on 2,4-dichlorophenoxyacetic acid (2,4-D) and arylophenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides). The protein have no metabolic role in plants (Dow AgroSciences, 2014). Expression level of AAD-1 in different plant parts of corn DAS-40278-9 was measured using ELISA methods (Dow AgroSciences, 2014). The measurements are in dry weight basis (ng/mg dry weight). Margin of exposure of general population and children in Japan to AAD-1 protein was derived from the data of the food intake in Japan and the No Observed Effect Level (NOEL) of AAD-1 protein determined from the acute oral toxicity study. Computed margins of exposure of general population and children (<6 year) to AAD-1 protein in corn is greater than 102564 and 67340, respectively. Toxicological and Allergenicity Assessment The novel protein, AAD-1, was subjected to digestibility, heat inactivation, oral toxicity and amino acid sequence comparison studies to determine its potential to cause toxicity or allergenicity to humans (Dow AgroSciences, 2014). Digestibility study using Simulated Gastric Fluid (SGF) with pepsin demonstrated that AAD-1 is readily degraded within 30 seconds of incubation with SGF, in presence of 0.32% w/v pepsin at pH 1.2, a characteristic of most non-toxic proteins (Dow AgroSciences, 2014). Heat Inactivation of AAD-1 is evaluated by heating protein solutions for 30 minutes at 50°C, 70°C and 95°C (Schafer, 2008). Upon treatment, samples were analyzed through ELISA, SDS-PAGE and western blot. Activity of AAD-1 was assayed by colorimetric enzyme assay. Results of the SDS-PAGE analysis showed that the AAD-1 protein was undetectable upon heating at 50, 70 or 95°C for 30 minutes. This was observed upon centrifugation of the protein samples prior to addition of Laemmli buffer. Immunoreactivity and enzymatic activity of AAD-1 decreased by 100% upon heat treatment at 50°C. Only 0.2% immunoreactivity and 3.0% enzymatic activity was detected upon heat treatment at 90°C. Amino acid sequence comparison of AAD-1 protein to toxins and allergens was conducted using BLASTp search algorithm against the GenBank and FASTA program (Dow AgroSciences, 2014). Results of bioinformatics analyses indicated that AAD-1 protein is not homologous to any toxin and allergen. This was verified through conducting amino acid comparison using the same bioinformatics tool on August 23, 2017 (AIS-FRA-17-06-BIA). Acute oral toxicity study on mice showed no mortality, clinical signs and treatment-related gross pathological observations during the study (Wiescinski and Golden, 2007). The determined No Observed Effect Level for AAD-1 protein is greater than 2000 mg/kg body weight. The AAD-1 protein used in the studies was obtained from Pseudomonas fluorescens (Dow AgroSciences, 2014). Biochemical characterization, SDS-PAGE and wester blot analysis of crude extracts, MALDI-TOF and ESI/LC-MS tryptic and Asp-N peptide mass fingerprints, tryptic and Asp-N Peptide N- and C- terminal sequence analysis, and endogenous allergen analysis were conducted by the proponent to confirm that the P. fluorescens- produced AAD-1 protein is biochemically and functionally equivalent to AAD-1 expressed in DAS-40278-9. Levels of AAD-1 in DAS-40278-9 determined through ELISA and the crude protein content of DAS-40278-9 were used to compute the percent total protein for AAD-1 which is 0.0046%. Results of the digestibility, heat inactivation, amino acid sequence comparison and acute oral toxicity studies indicates that AAD-1 protein being expressed in DAS-40278-9 corn is not toxic or allergenic to humans (Dow AgroSciences, 2014). Nutritional Data Compositional analysis provided by the developer indicating the nutritional data of DAS-40278-9 in comparison with the non-transgenic corn and range of literature values (Dow AgroSciences, 2014). The trials were conducted in Iowa, Illinois, Indiana, Nebraska and Ontario. Results of the analysis indicated that there is no differences in the proximate, fiber, mineral, amino acid, fatty acid, vitamins, anti-nutrient and secondary metabolite levels of DAS-40278-9 and the non-transgenic corn that can be considered biologically relevant. Conclusion For the transgenic DAS-40278-9 corn, enough evidence is provided to support the equivalence of the genetically modified crop, in terms of the nutritional composition and food safety, with the conventional corn other than tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides. After reviewing the provided material of Dow AgroSciences, it is therefore concluded that DAS-40278-9 corn is as safe as its conventional counterpart. BAI ASSESSMENT AND RECOMMENDATIONS Based on the documents submitted by the applicant, BAI made the following assessment: A. Host Organism Maize is a source of protein, amino acids, fatty acids, vitamins etc. It also contains low levels of several anti-nutrients including trypsin and chymotrypsin inhibitors, raffinose, phytic acid etc. Maize is consumed in various food forms including starch, oil, grits, meal and flour. Based on FAOSTAT data, consumption of maize intake by general population in ASEAN countries ranged from about 79-190g/person/day (Ranum et al., 2014) B. Transgenic Plant DAS 40278-9 is approved in: USA (USDA, 2014), Canada (CFIA, 2012; Health Canada, 2012), Brazil (CTNBio, 2015), Australia and New Zealand (FSANZ, 2011), Mexico (COFEPRIS, 2011), Columbia (ICA, 2013; INVIMA, 2014), South Africa (DAFF, 2012), Japan (MAFF, 2012; MHLW, 2012), Taiwan (DOH, 2011) and South Korea (MFDS, 2014; RDA, 2014). DAS-40278-9 maize is as safe and as nutritious as conventional maize. There is no need to change consumption pattern as a result of introduction of this Maize event. C. Donor Organism The AAD-1 protein is not known to possess potential pathogenic or allergenic properties. The introduced expressible sequence include AAD-1 protein conferring herbicide tolerance to 2,4dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides). Sphingobium herbicidovorans is a donor of AAD-1 protein. Donor organisms of genetic elements including promoters, terminators and border sequences include Nicotiana tobacum and Zea mays. There are no publications concerning toxicity or allergenicity of these genetic elements in peer-reviewed journals. AAD-1 is the only protein expressed in DAS-40278-9 maize. The protein has specific mode of action and have no significant sequence similarity to known allergens or toxins D. Transformation System DAS-40278-9 maize was generated through direct insertion of the DNA fragment from plasmid pDAS1740 via Whiskers-mediated transformation. The genetic modification was intended to express AAD-1 protein in maize plants, thus provide tolerance to 2,4dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides). The transformation protocol is described fully. The plasmid vector, pDAS1740 (including orientation and relative location of genetic elements), is described adequately. There is no carrier DNA used for the transformation of pDAS1740 into maize. Whiskers-mediated transformation is a direct DNA transfer method. E. Inserted DNA DAS-40278-9 maize contains one intact copy of the T-DNA insert at a single locus. The insert copy number was checked through Southern blot analysis. Integrity and order of genetic elements in DAS-40278-9 maize were demonstrated via Southern blot analysis. Southern blot analysis showed that DAS-40278-9 maize contains a single intact copy of the aad1 expression cassette integrated at a single locus. The T-DNA insert in DAS-40278-9 maize contains a single, intact copy of each of the expression cassette for aad-1 gene. No vector backbone sequences were detected in event DAS-40278-9. There were no re-arrangements of the cassette observed. There is no plasmid vector backbone sequence present in DAS-40278-9 maize as demonstrated by Southern blot analysis. Confirmation of lack of vector backbone using Southern blot analysis is a scientifically proven method and is sufficient. F. Genetic Stability Stability of the T-DNA insert across five generations was demonstrated by Southern blot analysis. Segregation was assessed using event-specific PCR. Populations of T1, T2 generations, 3 backcross populations (BC1, BC2, BC3) and one population of BC3F2 were assessed. Segregation result is consistent with the reported one copy TDNA insert. G. Expressed Material Expression levels of AAD-1 protein were determined using protein-specific ELISA methods with and without herbicide treatments. Expression values were similar for the sprayed treatments as well as for the plots sprayed and unsprayed with 2, 4-D and quizalofop herbicides. Mean AAD-1 protein levels in roots ranged from 2.87-3.92; in V9 stage leaf from 5.38-6.52; in forage from 6.84-7.32; in pollen from 108-127 and in grain from 4.61-5.00 ng/mg dry weight tissue. AAD-1 protein has specific mode of action on 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors (“fop” herbicides). The protein does not play a role in endogenous plant metabolism. H. Toxicological Assessment The digestibility of the microbe-derived AAD-1 protein was tested in vitro using simulated gastric fluid (SGF) containing gastric enzyme pepsin. The estimated T50 result was less than 30 seconds with no large size fragments remaining. AAD-1 protein was also evaluated by heating protein solutions for 30 min at 50, 70 and 95 C and 20 min in an autoclave in a phosphate based buffer. Results showed that AAD-1 protein is immunochemically denatured when heated. The AAD-1 protein lost more than 97% of its immunoreactivity, with results showing that it was almost undetectable by ELISA after exposure to the heat treatment. Bioinformatics analysis through BLAST search showed AAD-1 has no significant sequence similarity with known toxins (Section 9.1.3.1 of the Food and Feed safety and Nutrition Assessment for Herbicide Tolerance DAS-40278-9 Maize dossier). Glycosylation analysis of AAD-1 isolated from DAS-40278-9 maize showed the protein is not glycosylated.AAD-1 protein used in the acute toxicity test was derived from Pseudomonas fluorescens. The microbial AAD-1 is biochemically and functionally equivalent to the AAD-1 expressed in DAS-40278-9 I. Allergenicity Assessment The digestibility of the microbe-derived AAD-1 protein was tested in vitro using simulated gastric fluid (SGF) containing gastric enzyme pepsin. The estimated T50 result was less than 30 seconds with no large size fragments remaining. AAD-1 protein was also evaluated by heating protein solutions for 30 min at 50, 70 and 95 C and 20 min in an autoclave in a phosphate based buffer. Results showed that AAD-1 protein is immunochemically denatured when heated. The AAD-1 protein lost more than 97% of its immunoreactivity, with results showing that it was almost undetectable by ELISA after exposure to the heat treatment. Bioinformatics analysis through BLAST search showed AAD-1 has no significant sequence similarity with known toxins (Section 9.1.3.1 of the Food and Feed safety and Nutrition Assessment for Herbicide Tolerance DAS-40278-9 Maize dossier). Glycosylation analysis of AAD-1 isolated from DAS-40278-9 maize showed the protein is not glycosylated. When the WHO “GC 645 maize” acute consumption information is coupled to the AAD-1 field expression level of 4.81 ng/mg tissue, the potential acute exposure to AAD-1 protein via maize is estimated as: (1) 0.0195 mg protein/kg bw/day, for general population (i.e. adults) and (2) 0.0297 mg protein/kg bw/day, for children of 6 years or younger. No serum screening was performed for the said application. AAD-1 has no evidence of allergenicity. J. Nutritional Data Differences were observed in moisture and carbohydrates in grain between DAS-40278-9 maize and comparator. However, levels were all within reference range and literature range. Nutrient composition analysis showed DAS40278-9 maize is substantially equivalent to comparator, the non-transgenic Maize, with no significant and biologically meaningful differences, both in grains and forage. K. Recommendation Find scientific evidence that the regulated article applied for animal feed use is as safe as its conventional counterpart and shall not pose any significant risk to human and animal health DOH ASSESSMENT AND RECOMMENDATION After a thorough review and evaluation of the documents provided by the proponent, Dow AgroSciences B.V., Philippines Branch through the Bureau of Plant Industry (BPI), in support of their application for approval for Direct Use for Food and Feed or for Processing (FFP) of Corn DAS 40278-9. I/We, Find that the regulated article applied for Direct Use for Food and Feed or for Processing (FFP) is safe as its conventional counterpart and shall not pose any significant risk to human and animal health, and environment. The following are the observations and recommendations: 1) Find that the regulated article applied for Direct Use for FFP does not require changes in the usual practices in unloading and loading, hauling, transport and storage, and processing. As such, the regulated article is as safe as its conventional counterpart and is not expected to pose any significant risk to human and animal and the environment while in transit, storage and processing. 2) Scientific pieces of evidence from provided references i.e. literatures show that the regulated article applied for Direct Use as FFP is as safe as its conventional counterpart and shall not pose any significant risk to human and animal health and on the environment. 3) It is suggested that the BPI ensure the following: a) Strict monitoring of the regulated article from port of entry to the traders/importers storage/warehouse as stated in Sec 32 of JDC 1 s2016 b) The BPI to include in the issuance of permit for release of this product the following conditions: i. Any spillage (during unloading and loading/hauling and transport unloading and storage) shall be collected and cleaned up immediately. ii. Transportation of the consignment from the port of entry to any destination shall be in closed containers. iii. There shall be a clear instructions that the product is only for the purpose of direct use for FFP and is not to be used as planting materials. Based on the above considerations and with the submitted sworn statement and accountability of the proponent, this recommendation is being submitted to the BPI related to the processing and issuance of a biosafety permit for Direct Use as FFP of corn DAS 40278-9.

Please see the link below(in Korean).

Peer review of the data submitted by the applicant and the results of complex medical and biological studies of transgenic maize line DAS-40278-9 tolerant to 2,4-D and aryloxyphenoxypropionate herbicides, attest to the absence of any toxic, reprotoxic, genotoxic, or allergenic effects of this maize line. By biochemical composition, transgenic maize line DAS-40278-9 was identical to conventional maize. GM maize line DAS-40278-9 has been registered for food use, listed in the State Register, and licensed for use in the territory of The Eurasian Economic Union, import into the territory of The Eurasian Economic Union, and placing on the market without restrictions.

Herbicide-tolerant corn line DAS-40278-9 has been genetically modified to tolerate 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) acetyl coenzyme A carboxylase (ACCase) inhibitors such as quizalofop-P-ethyl. This has been achieved through the introduction of the aad-1 gene, from Sphingobium herbicidovorans, expressing the enzyme aryloxyalkanoate dioxygenase (AAD-1). Molecular analyses of corn DAS-40278-9 indicate there is one insertion site at a single genetic locus. This site contains one copy of the aad-1 gene and it is stably inherited from one generation to the next generation. Corn DAS-40278-9 expresses one new protein, AAD-1, which is non-toxic and non-allergenic to humans. Composition analyses showed that the levels of key components in seed from corn DAS-40278-9 are equivalent to conventional corn cultivars. Food derived from corn DAS-40278-9 is as safe as food derived from conventional corn cultivars.

The GM maize DAS-40278-9 has been assessed in terms of the Genetically Modified Organisms Act, 1997 by the Advisory Committee, a scientific panel and the Executive Council an intergovernmental decision making body. The assessment considered amongst others the following: The source of the gene, nature of host organism, protein expression, toxicology and allergenicity issues

Corn
Trait 1 Added Protein: Aryloxyalkanoate dioxygenase-1 (AAD-1) protein encoded by the aad-1 gene
Source: Sphingobium herbicidovorans
Intended Effect: Tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and certain aryloxyphenoxypropionate herbicides (e.g., quizalofop, cyhalofop, haloxyfop)

Please consult the FDA website links below.