Inkoo and Sindbis viruses in blood sucking insects, and a serological study for Inkoo virus in semi-domesticated Eurasian tundra reindeer in Norway.

BACKGROUND: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. MATERIALS AND METHODS: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013-2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). RESULTS: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). CONCLUSION: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norway indicate that further studies are required for monitoring this virus. SINV was not detected in the BSI in this study, however, human cases of SINV infection are yearly reported from other regions such as Rjukan in south-central Norway. It is therefore essential to monitor both viruses in the human population. Our findings are important to raise awareness regarding the geographical distribution of these mosquito-borne viruses in Northern Europe.

Serological screening for tick-borne encephalitis virus in eight Norwegian herds of semi-domesticated reindeer (Rangifer tarandus tarandus).

Tick-borne encephalitis virus (TBEV) is found in Ixodes ricinus ticks throughout the area where viable tick populations exist. In Norway, TBEV is found in I. ricinus from the south coast until Brønnøy municipality in Nordland County and the range of the vector is expanding due to changes in climate, vegetation, host animals and environmental conditions. TBEV might thus have the potential to establish in new areas when I. ricinus expand its geographical distribution. At present, there is little knowledge on the status of the virus in high-altitude areas of inland regions in Norway. It has previously been indicated that reindeer may be an important sentinel species and indicator of the spread of ticks and TBEV in high-altitude regions. In this study, 408 semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) from eight herds, from Tana in Troms and Finnmark County in northern Norway to Filefjell in Innlandet and Viken Counties in southern Norway, were screened for TBEV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We found 16 TBEV reactive reindeer samples by ELISA; however, these results could not be confirmed by the serum neutralization test (SNT). This could indicate that a flavivirusand not necessarily TBEV, may be circulating among Norwegian semi-domesticated reindeer. The results also indicate that TBEV was not enzootic in Norwegian semi-domesticated reindeer in 2013-2015. This knowledge is important as an information base for future TBEV and flavivirus surveillance in Norway.

Prevalence of tick-borne encephalitis virus in questing Ixodes ricinus nymphs in southern Scandinavia and the possible influence of meteorological factors.

Ixodes ricinus ticks are Scandinavia's main vector for tick-borne encephalitis virus (TBEV), which infects many people annually. The aims of the present study were (i) to obtain information on the TBEV prevalence in host-seeking I. ricinus collected within the Øresund-Kattegat-Skagerrak (ØKS) region, which lies in southern Norway, southern Sweden and Denmark; (ii) to analyse whether there are potential spatial patterns in the TBEV prevalence; and (iii) to understand the relationship between TBEV prevalence and meteorological factors in southern Scandinavia. Tick nymphs were collected in 2016, in southern Scandinavia, and screened for TBEV, using pools of 10 nymphs, with RT real-time PCR, and positive samples were confirmed with pyrosequencing. Spatial autocorrelation and cluster analysis was performed with Global Moran's I and SatScan to test for spatial patterns and potential local clusters of the TBEV pool prevalence at each of the 50 sites. A climatic analysis was made to correlate parameters such as minimum, mean and maximum temperature, relative humidity and saturation deficit with TBEV pool prevalence. The climatic data were acquired from the nearest meteorological stations for 2015 and 2016. This study confirms the presence of TBEV in 12 out of 30 locations in Denmark, where six were from Jutland, three from Zealand and two from Bornholm and Falster counties. In total, five out of nine sites were positive from southern Sweden. TBEV prevalence of 0.7%, 0.5% and 0.5%, in nymphs, was found at three sites along the Oslofjord (two sites) and northern Skåne region (one site), indicating a potential concern for public health. We report an overall estimated TBEV prevalence of 0.1% in questing I. ricinus nymphs in southern Scandinavia with a region-specific prevalence of 0.1% in Denmark, 0.2% in southern Sweden and 0.1% in southeastern Norway. No evidence of a spatial pattern or local clusters was found in the study region. We found a strong correlation between TBEV prevalence in ticks and relative humidity in Sweden and Norway, which might suggest that humidity has a role in maintaining TBEV prevalence in ticks. TBEV is an emerging tick-borne pathogen in southern Scandinavia, and we recommend further studies to understand the TBEV transmission potential with changing climate in Scandinavia.

Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana.

Flaviviruses are a threat to public health and can cause major disease outbreaks. Tick-borne encephalitis (TBE) is caused by a flavivirus, and it is one of the most important causes of viral encephalitis in Europe and is on the rise in Sweden. As there is no antiviral treatment available, vaccination remains the best protective measure against TBE. Currently available TBE vaccines are based on formalin-inactivated virus produced in cell culture. These vaccines must be delivered by intramuscular injection, have a burdensome immunization schedule, and may exhibit vaccine failure in certain populations. This project aimed to develop an edible TBE vaccine to trigger a stronger immune response through oral delivery of viral antigens to mucosal surfaces. We demonstrated successful expression and post-translational processing of flavivirus structural proteins which then self-assembled to form virus-like particles in Nicotiana benthamiana. We performed oral toxicity tests in mice using various plant species as potential bioreactors and evaluated the immunogenicity of the resulting edible vaccine candidate. Mice immunized with the edible vaccine candidate did not survive challenge with TBE virus. Interestingly, immunization of female mice with a commercial TBE vaccine can protect their offspring against TBE virus infection.

Diversity of Rotavirus Strains in Children; Results From a Community-Based Study in Nepal.

Objective: The objectives of this study were to describe the incidence and genetic diversity of Rotavirus (RV) infection among children up to 3 years of age in a community in Nepal. Methods: We investigated community-acquired cases of asymptomatic and symptomatic RV infections in children from birth to 36 months of age in a community-based birth cohort in Bhaktapur, Nepal. Monthly surveillance and diarrheal stool samples were collected from 240 children enrolled at birth, of which 238 completed the 3 years of follow-up. Samples were screened for rotavirus by Enzyme Immuno Assay (EIA). All RV screened positives were further genotyped by reverse transcription-polymerase chain reaction for the capsid genes VP7 and VP4. Results: In total, 5,224 stool samples were collected from 238 children, followed from birth to 36 months of age. Diarrhea occurred in 92.4% (230/238) of all children in the cohort. During the 3 years study period, RV was more frequently seen in children with symptoms (7.6%) than in non-symptomatic children (0.8%). The highest RV detection rate was found in younger children between 3 and 21 months of age. Although rotavirus is known as winter diarrhea, it was detected throughout the year except in August. The highest positivity rate was observed in the months between December and March, with a peak in January. Four common G types were seen: G2 (30%), G1 (29%), G12 (19%), and G9 (16%). The most predominant genotypes seen were G2P[4] (30%), followed by G1P[8] (27.0%), G12P[6] (14.0%), G9P[8] (10%), and remaining were mixed, partial, and untyped. Conclusion: Our study confirms that rotavirus is a common cause of gastroenteritis in young children in the community. The prevalence and pathogenicity of rotavirus infection differed by age. There was substantial variability in circulating strains in the community samples compared to samples collected from hospitals. This shows the importance of including community-based surveillance systems to monitor the diversity of circulating rotavirus strains in Nepal.

High-throughput sequencing of two European strains of tick-borne encephalitis virus (TBEV), Hochosterwitz and 1993/783.

Tick-borne encephalitis virus (TBEV) is a medically important arbovirus, widespread in Europe and Asia. The virus is primarily transmitted to humans and animals by bites from ticks and, in rare cases, by consumption of unpasteurized dairy products. The aim of this study was to sequence and characterize two TBEV strains with amplicon sequencing by designing overlapping primers. The amplicon sequencing, via Illumina MiSeq, covering nearly the entire TBEV genome, was successful: We retrieved and characterized the complete polyprotein sequence of two TBEV strains, Hochosterwitz and 1993/783 from Austria and Sweden, respectively. In this study the previous phylogenetic analysis of both strains was confirmed to be of the European subtypes of TBEV (TBEV-Eu) by whole genome sequencing. The Hochosterwitz strain clustered with the two strains KrM 93 and KrM 213 from South Korea, and the 1993/783 strain clustered together with the NL/UH strain from the Netherlands. Our study confirms the suitability and rapidness of the high-throughput sequencing method used to produce complete TBEV genomes from TBEV samples of high viral load giving high-molecular-weight cDNA with large overlapping amplicons.

Cervids as sentinel-species for tick-borne encephalitis virus in Norway - A serological study.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE). TBEV is one of the most important neurological pathogens transmitted by tick bites in Europe. The objectives of this study were to investigate the seroprevalence of TBE antibodies in cervids in Norway and the possible emergence of new foci, and furthermore to evaluate if cervids can function as sentinel animals for the distribution of TBEV in the country. Serum samples from 286 moose, 148 roe deer, 140 red deer and 83 reindeer from all over Norway were collected and screened for TBE immunoglobulin G (IgG) antibodies with a modified commercial enzyme-linked immunosorbent assay (ELISA) and confirmed by TBEV serum neutralisation test (SNT). The overall seroprevalence against the TBEV complex in the cervid specimens from Norway was 4.6%. The highest number of seropositive cervids was found in south-eastern Norway, but seropositive cervids were also detected in southern- and central Norway. Antibodies against TBEV detected by SNT were present in 9.4% of the moose samples, 1.4% in red deer, 0.7% in roe deer, and nil in reindeer. The majority of the positive samples in our study originated from areas where human cases of TBE have been reported in Norway. The study is the first comprehensive screening of cervid species in Norway for antibodies to TBEV, and shows that cervids are useful sentinel animals to indicate TBEV occurrence, as supplement to studies in ticks. Furthermore, the results indicate that TBEV might be spreading northwards in Norway. This information may be of relevance for public health considerations and supports previous findings of TBEV in ticks in Norway.

Geographical distribution and prevalence of tick-borne encephalitis virus in questing Ixodes ricinus ticks and phylogeographic structure of the Ixodes ricinus vector in Norway.

The tick-borne encephalitis virus (TBEV), a zoonotic flaviviral infection, is endemic in large parts of Norway and Eurasia. Humans are mainly infected with TBEV via bites from infected ticks. In Norway, the main geographical distribution of ticks is along the Norwegian coastline from southeast (~59°N) and up to the southern parts of Nordland County (~65°N). In this study, we collected ticks by flagging along the coast from Østfold County to Nordland County. By whole-genome sequencing of the mitochondrial genome of Ixodes ricinus, the phylogenetic tree suggests that there is limited phylogeographic structure both in Norway and in Europe. The overall TBEV prevalence is 0.3% for nymphs and 4.3% for adults. The highest estimated TBEV prevalence in adult ticks was detected in Rogaland and Vestfold County, while for nymphs it is highest in Vestfold, Vest-Agder and Rogaland. The present work is one of the largest studies on distribution and prevalence of TBEV in ticks in Scandinavia, showing that the virus is wider distributed in Norway than previously anticipated.

Experimental infection of lambs with tick-borne encephalitis virus and co-infection with Anaplasma phagocytophilum.

Tick-borne encephalitis virus (TBEV) is a zoonotic pathogen which may cause tick-borne encephalitis (TBE) in humans and animals. More than 10,000 cases of TBE are reported annually in Europe and Asia. However, the knowledge on TBE in animals is limited. Co-infection with Anaplasma phagocytophilum and louping ill virus (LIV), a close relative to TBEV, in sheep has been found to cause more severe disease than single LIV or A. phagocytophilum infection. The aim of this study was to investigate TBEV infection and co-infection of TBEV and A. phagocytophilum in lambs. A total of 30 lambs, aged five to six months, were used. The experiment was divided into two. In part one, pre- and post-infection of TBEV and A. phagocytophilum was investigated (group 1 to 4), while in part two, co-infection of TBEV and A. phagocytophilum was investigated (group 5 and 6). Blood samples were drawn, and rectal temperature was measured daily. Lambs inoculated with TBEV displayed no clinical symptoms, but had a short or non-detectable viremia by reverse transcription real-time PCR. All lambs inoculated with TBEV developed neutralizing TBEV antibodies. Our study is in accordance with previous studies, and indicates that TBEV rarely causes symptomatic disease in ruminants. All lambs inoculated with A. phagocytophilum developed fever and clinical symptoms of tick-borne fever, and A. phagocytophilum was present in the blood samples of all infected lambs, shown by qPCR. Significantly higher mean TBEV titer was detected in the group co-infected with TBEV and A. phagocytophilum, compared to the groups pre- or post-infected with A. phagocytophilum. These results indicate that co-infection with TBEV and A. phagocytophilum in sheep stimulates an increased TBEV antibody response.

Tick-borne encephalitis virus in cows and unpasteurized cow milk from Norway.

Tick-borne encephalitis virus (TBEV) is recognized as the most important zoonotic tick-transmitted virus in Europe. TBEV is mainly transmitted to humans through bites from TBEV-infected ticks (Ixodes ricinus and Ixodes persulcatus). However, alimentary infection after consumption of unpasteurized milk and cheese from domestic ruminants has been reported. There is little information about TBEV in ruminants in Norway. The objectives of this study were to analyse unpasteurized cow milk for TBEV RNA and to study the presence of IgG antibodies to TBEV in the same animals. A total of 112 milk and blood samples were collected from cows from five different farms spread from southern to northern Norway. The milk samples were analysed by an in-house reverse transcription (RT) real-time polymerase chain reaction and confirmed by pyrosequencing. Serum samples were screened by a commercial enzyme-linked immunosorbent assay and verified by a TBEV-specific serum neutralization test. We found TBEV RNA in unpasteurized milk collected from farms in the municipalities of Mandal, Skedsmo and Brønnøy in 5.4% of the tested animals. Specific antibodies to TBEV were only detected in Arendal, where 88.2% of the tested animals were positive. Further studies on milk containing TBEV RNA should be performed to conclude if TBEV found in unpasteurized milk in Norway is infectious, which could be of great importance in a One Health perspective.

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene.

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.

Adenomatous polyposis coli truncation mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumours of multiple intestinal neoplasia mice.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces intestinal tumours in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. The main mechanism for PhIP-induced tumour induction in Min/+ mice is loss of the wild-type adenomatous polyposis coli (Apc) allele, i.e. loss of heterozygosity (LOH). In this study, single injections of either 10, 17.5 or 25 mg/kg PhIP on days 3-6 after birth all increased the mean number of small intestinal tumours two to three-fold, from 37.7 in controls to 124.8 in the PhIP-treated Min/+ mice. In total, we analysed 292 small intestinal tumours and 253 of these had LOH. The frequency of LOH in the Apc gene was 88, 93, 83 and 84% in tumours of 0, 10, 17.5 and 25 mg/kg PhIP-treated mice, respectively. Therefore, these lower doses of PhIP did not reduce the frequency of LOH, as found in our previous study with a single injection of 50 mg/kg PhIP (Mutat. Res. 1-2 (2002) 157). In the second part of this study, we wanted to characterise Apc truncation mutations from tumour samples apparently retaining the Apc wild-type allele from this and two previous experiments with PhIP-exposed Min/+ mice. In the first half of exon 15 in Apc, we verified 25 mutations from 804 tumour samples of PhIP-treated mice. Of these were 60% G-->T transversions, and 16% G deletions, indicating that these are the predominant types of PhIP-induced truncation mutations in the Apc gene in Min/+ mice. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the beta-catenin binding region. We also identified two Apc truncation mutations from 606 spontaneously formed intestinal tumours from untreated Min/+ mice, one C-->T transition and one T insertion, which were different from those induced by PhIP.

Environmental Risk Assessment of Glufosinate-Tolerant Genetically Modified Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Scientific Committee for Food Safety (VKM) has been requested by the Norwegian Directorate for Nature Management to conduct final environmental risk assessments for all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC. The request covers scope(s) relevant to the Gene Technology Act. The request does not cover GMOs that VKM already has conducted its final risk assessments on. However, the Directorate requests VKM to consider whether updates or other changes to earlier submitted assessments are necessary. The genetically modified, glufosinate-tolerant oilseed rape lines MS8, RF3 and MS8 x RF3 (Notification C/BE/96/01) are approved under Directive 2001/18/EC for import and processing for feed and industrial purposes since 26 March 2007 (Commission Decision 2007/232/EC). In addition, processed oil from genetically modified oilseed rape derived from MS8, RF3 and MS8 x RF3 were notified as existing food according to Art. 5 of Regulation (EC) No 258/97 on novel foods and novel food ingredients in November 1999. Existing feed and feed products containing, consisting of or produced from MS8, RF3 and MS8 x RF3 were notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003 and were placed on the market in January 2000. An application for renewal of the authorisation for continued marketing of existing food, food ingredients and feed materials produced from MS8, RF3 and MS8 x RF3 was submitted within the framework of Regulation (EC) No 1829/2003 in June 2007 (EFSA/GMO/RX/MS8/RF3). In addition, an application covering food containing or consisting of, and food produced from or containing ingredients produced from oilseed rape MS8, RF3 and MS8 x RF3 (with the exception of processed oil) was delivered by Bayer CropScience in June 2010 (EFSA/GMO/BE/2010/81). The VKM GMO Panel has previously issued a scientific opinion related to the notification C/BE/96/01 for the placing on the market of the oilseed rape lines for import, processing and feed uses (VKM 2008). The health and environmental risk assessment was commissioned by the Norwegian Directorate for Nature Management in connection with the national finalisation of the procedure of the notification C/BE/96/01 in 2008. Due to the publication of updated guidelines for environmental risk assessments of genetically modified plants and new scientific literature, the VKM GMO Panel has decided to deliver an updated environmental risk assessment of oilseed rape MS8, RF3 and MS8 x RF3. A scientific opinion on an application for the placing on the market of MS8/RF3 for food containing or consisting of, and food produced from or containing ingredients produced from MS8/RF3 (with the exception of processed oil) (EFSA/GMO/BE/2010/81) have also been submitted by the VKM GMO Panel (VKM 2012). The environmental risk assessment of the oilseed rape MS8, RF3 and MS8 x RF3 is based on information provided by the notifier in the applications EFSA/GMO/RX/MS8/RF3, EFSA/GMO/BE/2010/8, the notification C/BE/96/01, and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated MS8, RF3 and MS8 x RF3 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2006, 2011a), the environmental risk assessment of GM plants (EFSA 2010), the selection of comparators for the risk assessment of GM plants (EFSA 2011b), and for the post-market environmental monitoring of GM plants (EFSA 2006, 2011c). The scientific risk assessment of oilseed rape MS8, RF3 and MS8 x RF3 include molecular characterisation of the inserted DNA and expression of target proteins, comparative assessment of agronomic and phenotypic characteristics, unintended effects on plant fitness, potential for horizontal and vertical gene transfer, and evaluations of the post-market environmental plan. In line with its mandate, VKM emphasised that assessments of sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act, shall not be carried out by the Panel on Genetically Modified Organisms. The genetically modified oilseed rape lines MS8 and RF3 were developed to provide a pollination control system for production of F1-hybrid seeds (MS8 x RF3). Oilseed rape is a crop capable of undergoing both self-pollination (70%) as well as cross-pollination (30%). Therefore a system to ensure only cross-pollination is required for producing hybrids from two distinct parents. As a result of hybrid vigor cross-pollinated plants produce higher yield as compared to self-pollinating rape. The hybrid system is achieved using a pollination control system by insertion and expression of barnase and barstar genes derived from the soil bacterium Bacillus amyloliquefaciens into two separate transgenic oilseed rape lines. The barnase gene in the male sterile line MS8 encode a ribonuclease peptide (RNase), expressed in the tapetum cells during anther development. The RNase effect RNA levels, disrupting normal cell function, arresting early anther development, and results in the lack of viable pollen and male sterility. The fertility restoration line RF3 contains a barstar gene, coding for a ribonuclease inhibitor (Barstar peptide) expressed only in the tapetum cells of the pollen during anther development. The peptide specifically inhibits the Barnase RNase expressed by the MS8 line. The RNase and the ribonuclease inhibitor form a stable one-to-one complex, in which the RNase is inactivated. As a result, when pollen from the receptor line RF3 is crossed to the male sterile line MS8, the MS8 x RF3 progeny expresses the RNase inhibitor in the tapetum cells of the anthers allowing hybrid plants to develop normal anthers and restore fertility. The barnase and barstar genes in MS8 and RF3 are each linked with the bar gene from Streptomyces hygroscopus. The bar gene is driven by a plant promoter that is active in all green tissues of the plant, and encodes the enzyme phosphinothricin acetyltransferase (PAT). The PAT enzyme inactivates phosphinothricin (PPT), the active constituent of the non-selective herbicide glufosinate-ammonium. The bar gen were transferred to the oilseed rape plants as markers both for use during in vitro selection and as a breeding selection tool in seed production. Molecular characterization: The oilseed rape hybrid MS8xRF3 is produced by conventional crossing. The parental lines MS8 and RF3 are well described in the documentation provided by the applicant, and a number of publications support their data. It seems likely that MS8 contains a complete copy of the desired T-DNA construct including the bar and barnase genes. Likewise, the event RF3 is likely to contain complete copies of the bar and barstar genes in addition to a second incomplete non-functional copy of the bar-gene. The inserts in the single events are preserved in the hybrid MS8xRF3, and the desired traits are stably inherited over generations. Oilseed rape MS8, RF3 and MS8xRF3 and the physical, chemical and functional characteristics of the newly expressed proteins have previously been evaluated by the VKM Panel on Genetically Modified Organisms, and considered satisfactory (VKM 2008, 2012). The GMO Panel finds the characterisation of the physical, chemical and functional properties of the recombinant inserts in the oilseed rape transformation events MS8, RF3 and MS8xRF3 to be satisfactory. The GMO Panel has not identified any novel risks associated with the modified plants based on the molecular characterisation of the inserts. Comparative assessment: Based on results from comparative analyses of data from field trials located at representative sites and environments in Europe and Canada, it is concluded that oilseed rape MS8, RF3 and MS8 x RF3 is agronomically and phenotypically equivalent to the conventional counterpart, except for the newly expressed barnase, barstar and PAT proteins. The field evaluations support a conclusion of no phenotypic changes indicative of increased plant weed/pest potential of event MS8, RF3 and MS8 x RF3 compared to conventional oilseed rape. Furthermore, the results demonstrate that in-crop applications of glufosinate herbicide do not alter the phenotypic and agronomic characteristics of event MS8, RF3 and MS8 x RF3 compared to conventional oilseed rape varieties. Environmental risk: Considering the scope of the notification C/BE/96/01, excluding cultivation purposes, the environmental risk assessment is limited to exposure through accidental spillage of viable seeds of MS8, RF3 and MS8 x RF3 into the environment during transportation, storage, handling, processing and use of derived products. Oilseed rape is mainly a self-pollinating species, but has entomophilous flowers capable of both self- and cross-pollinating. Normally the level of outcrossing is about 30%, but outcrossing frequencies up to 55% are reported. Several plant species related to oilseed rape that are either cultivated, occurs as weeds of cultivated and disturbed lands, or grow outside cultivation areas t

Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON810 for Cultivation, Seed Production, Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/F/95/12/02)

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Scientific Committee for Food Safety (VKM) has been requested by the Norwegian Directorate for Nature Management to conduct final environmental risk assessments for all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC. The request covers scope(s) relevant to the Gene Technology Act. The request does not cover GMOs that VKM already has conducted its final risk assessments on. However, the Directorate requests VKM to consider whether updates or other changes to earlier submitted assessments are necessary. MON810 notification C/F/95/12-02 is approved under Directive 90/220/EEC for cultivation, seed production, import and processing into feeding stuffs and industrial purposes since 22 April 1998 (Commission Decision 98/294/EC). In December 1997, food and food ingredients derived from the progeny of maize line MON810 were notified under Article 5 of Regulation (EC) No 258/97 on novel foods and novel food ingredients. In addition, existing food and feed products containing, consisting of or produced from MON810 were notified according to Articles 8 and 20 of Regulation (EC) No 1829/2003 and were placed in the Community Register in 2005. Three applications for renewal of the authorisation for continued marketing of (1) existing food and food ingredients produced from MON810; (2) feed consisting of and/or containing maize MON810, and MON810 for feed use (including cultivation); and (3) food and feed additives, and feed materials produced from maize MON810 within the framework of Regulation (EC) No 1829/2003 were submitted in 2007. Maize MON810 has previously been assessed by the VKM GMO Panel commissioned by the Norwegian Directorate for Nature Management in connection with the national finalisation of the procedure of the notification C/F/95/12/02 (VKM 2007a,b). In addition, MON810 has been evaluated by the VKM GMO Panel as a component of several stacked GM maize events (VKM 2005a,b,c, VKM 2007c, VKM 2008, VKM 2009, VKM 2012). Due to the publication of updated guidelines for environmental risk assessments of genetically modified plants and new scientific literature, the VKM GMO Panel has decided to deliver an updated environmental risk assessment of MON810. The environmental risk assessment of the maize MON810 is based on information provided by the applicant in the notification C/F/95/12/02 and application EFSA/GMO/RX/MON810, and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated MON810 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2006, 2011a), the environmental risk assessment of GM plants (EFSA 2010), the selection of comparators for the risk assessment of GM plants (EFSA 2011b), and for the post-market environmental monitoring of GM plants (EFSA 2006, 2011c). The scientific risk assessment of maize MON810 include molecular characterisation of the inserted DNA and expression of the target protein, comparative assessment of agronomic and phenotypic characteristics, unintended effects on plant fitness, potential for gene transfer, interactions between the GM plant and target and non-target organisms, effects on biogeochemical processes and evaluations of the post-market environmental plan. In line with its mandate, VKM emphasised that assessments of sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act, shall not be carried out by the Panel on Genetically Modified Organisms. The genetically modified maize MON810 was developed to provide protection against certain lepidopteran target pests, including European corn borer (Ostrinia nubilalis) and species belonging to the genus Sesamia. Protection is achieved through expression in the plant of the insecticidal Cry protein, Cry1Ab, derived from Bacillus thuringiensis ssp. kurstaki, a common soil bacterium. Molecular characterisation Appropriate analysis of the integration site including flanking sequences and bioinformatics analyses have been performed to analyse the construct integrated in the GM plant. Updated bioinformatics analyses revealed that one ORF shared sequence similarity to a putative HECT-ubiquitin ligase protein. The VKM GMO Panel found no safety implications from the interruption of this gene sequence. Analyses of leaf, grains, whole plant tissue and pollen from the maize MON 810 demonstrated that the Cry1Ab protein is expressed at very low levels in all tissues tested and constitutes less than 0.001% of the fresh weight in each tissue. The cry1Ab gene is the only transgene expressed in line MON 810 and was expressed highest in the leaves. The stability of the genetic modification has been demonstrated over several generations. Event MON810 and the physical, chemical and functional characteristics of the proteins have previously been evaluated by The VKM Panel on Genetically Modified Organisms, and considered satisfactory (VKM 2007a,b). Comparative assessment: Comparative analyses of data from field trials located at representative sites and environments in the USA and Europe indicate that maize MON810 is agronomically and phenotypically equivalent to the conventional counterpart and commercially available reference varieties, with the exception of the lepidopteran-protection trait, conferred by the expression of the Cry1Ab protein. The field evaluations support a conclusion of no phenotypic changes indicative of increased plant weed/pest potential of MON810 compared to conventional maize. Evaluations of ecological interactions between maize MON810 and the biotic and abiotic environment indicate no unintended effects of the introduced trait on agronomic and phenotypic characteristics. Environmental risk: There are no reports of the target lepidopteran species attaining pest status on maize in Norway. Since there are no Bt-based insecticides approved for use in Norway, and lepidopteran pests have not been registered in maize, issues related to resistance evolution in target pests are not relevant at present for Norwegian agriculture. Published scientific studies show no or negligible adverse effects of Cry1Ab protein on non-target arthropods that live on or in the vicinity of maize plants. Cultivation of maize MON810 is not considered to represent a threat to the prevalence of red-listed species in Norway. Few studies have been published examining potential effects of Cry1Ab toxin on ecosystems in soil, mineralization, nutrient turnover and soil communities. Some field studies have indicated that root exudates and decaying plant material containing Cry proteins may affect population size and activity of rhizosphere organisms (soil protozoa and microorganisms). Most studies conclude that effects on soil microorganisms and microbial communities are transient and minor compared to effects caused by agronomic and environmental factors. However, data are only available from short term experiments and predictions of potential long term effects are difficult to deduce. Few studies have assessed the impact of Cry proteins on non-target aquatic arthropods and the fate of these proteins in senescent and decaying maize detritus in aquatic environments. However, exposure of non-target organisms to Cry proteins in aquatic ecosystems is likely to be very low, and potential exposure of Bt toxins to non-target organisms in aquatic ecosystems in Norway is considered to be negligible. Maize is the only representative of the genus Zea in Europe, and there are no cross-compatible wild or weedy relatives outside cultivation with which maize can hybridise and form backcross progeny. Vertical gene transfer in maize therefore depends on cross-pollination with other conventional or organic maize varieties. In addition, unintended admixture of genetically modified material in seeds represents a possible way for gene flow between different crop cultivations. The risk of pollen flow from maize volunteers is negligible under Norwegian growing conditions. In addition to the data presented by the applicant, the VKM GMO Panel is not aware of any scientific report of increased establishment and spread of maize MON810 and any change in survival (including over-wintering), persistence and invasiveness capacity. Because the general characteristics of maize MON810 are unchanged, insect resistance are not likely to provide a selective advantage outside cultivation in Norway. Since MON810 has no altered agronomic and phenotypic characteristics, except for the specific target pest resistance, the VKM GMO Panel is of the opinion that the likelihood of unintended environmental effects due to the establishment and survival of maize MON810 will be no different to that of conventional maize varieties in Norway. Overall conclusion: The VKM GMO Panel concludes that cultivation of maize MON810 is unlikely to have any adverse effect on the environment in Norway.

Environmental Risk Assessment of Glufosinate-Tolerant Genetically Modified Oilseed Rape T45 for Food and Feed Uses, Import and Processing under Regulation (EC) No 1829/2003 (Application EFSA/GMO/UK/2005/25)

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Scientific Committee for Food Safety (VKM) has been requested by the Norwegian Directorate for Nature Management to conduct final environmental risk assessments for all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC. The assignment includes a scientific environmental risk assessment of oilseed rape T45 (Reference EFSA/GMO/UK/2005/25) from Bayer CropScience for food and feed uses, import and processing. Oilseed rape T45 has previously been risk assessed by the VKM Panel on Genetically Modified Organisms (GMO), commissioned by the Norwegian Food Safety Authority related to the EFSAs public hearing in 2007 (VKM 2007a). Food additives produced from T45 oilseed rape were notified in the EU as existing food additives within the meaning of Article 8 (1)(b) of Regulation 1829/2003, authorized under Directive 89/10/EEC (Community Register 2005). Feed materials produced from T45 were also notified as existing feed products containing, consisting of or produced from T45 according to Articles 8 and 20 of Regulation (EC) No 1829/2003 in 2003. A notification for placing on the market of T45 according to the Directive 2001/18/EC was submitted in March 2004 (C/GB/04/M5/4), covering import and processing of T45 into food and feed. The application was further transferred into Regulation (EC) No 1829/2003 in November 2005 (EFSA/GMO/UK/2005/25). An application for renewal of authorisation for continued marketing of food additives and feed materials produced from T45 oilseed rape was submitted under Regulation (EC) No 1829/2003 in 2007 (EFSA/GMO/RX/T45). The EFSA GMO Panel performed one single comprehensive risk assessment for all intended uses of genetically modified oilseed rape T45, and issued a comprehensive scientific opinion for both applications submitted under Regulation (EC) No 1829/2003. The scientific opinion was published in January 30 2008 (EFSA 2008), and food and feed products containing or produced from oilseed rape T45 was approved by Commission Decision 26 March 2009 (Commission Decision 2009/184/EC). The oilseed rape T45 is however currently being phased out (EU-COM 2009). The commercialisation of T45 oilseed rape seeds in third countries was stopped after the 2005 planting season and stocks of all oilseed rape T45 lines have been recalled from distribution and destroyed. The applicant commits not to commercialize the event in the future and the import will therefore be restricted to adventitious levels in oilseed rape commodity. Thus the incidence of oilseed rape T45 in the EU is expected to be limited. The environmental risk assessment of the oilseed rape T45 is based on information provided by the notifier in the application EFSA/GMO/UK/2005/25 and EFSA/GMO/RX/T45, and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated T45 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2006, 2011a), the environmental risk assessment of GM plants (EFSA 2010), the selection of comparators for the risk assessment of GM plants (EFSA 2011b), and for the post-market environmental monitoring of GM plants (EFSA 2006, 2011c). The scientific risk assessment of oilseed rape T45 include molecular characterisation of the inserted DNA and expression of target proteins, comparative assessment of agronomic and phenotypic characteristics, unintended effects on plant fitness, potential for horizontal and vertical gene transfer, and evaluations of the post-market environmental plan. In line with its mandate, VKM emphasised that assessments of sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act, shall not be carried out by the Panel on Genetically Modified Organisms. The glufosinate ammonium-tolerant oilseed rape transformation event T45 (Unique Identfier ACSBNØØ8-2) was developed by Agrobacterium-mediated transformation of protoplast from the conventional oilseed rape cultivar “AC Excel”. T45 contains a synthetic version of the native pat gene isolated from the bacteria Streptomyces viridochromogenes, strain Tü 494. The inserted gene encodes the enzyme phosphinothricin acetyltransferase (PAT), which confers tolerance to the herbical active substance glufosinate ammonium. The PAT enzyme detoxifies glufosinate-ammonium by acetylation of the L-isomer into N-acetyl-L-glufosinate ammonium (NAG) which does not inhibit glutamine synthetase and therefore confers tolerance to the herbicide. Glufosinate ammonium-tolerant oilseed rape transformation event T45 has been conventionally bred into an array of spring-type oilseed rape varieties. Molecular characterization: The molecular characterisation data established that only one copy of the gene cassette is integrated in the oilseed rape genomic DNA. Appropriate analysis of the integration site including sequence determination of the inserted DNA and flanking regions and bioinformatics analysis have been performed. Bioinformatics analyses of junction regions demonstrated the absence of any potential new ORFs coding for known toxins or allergens. The genetic stability of transformation event T45 was demonstrated at the genomic level over multiple generations by Southern analysis. Segregation analysis shows that event T45 is inherited as dominant, single locus trait. Phenotypic stability has been confirmed by stable tolerance to the herbicide for T45 lines and varieties derived from the event grown in Canada since 1993. Oilseed rape transformation event T45 and the physical, chemical and functional characteristics of the proteins have previously been evaluated by The VKM Panel on Genetically Modified Organisms, and considered satisfactory (VKM 2007a). Comparative assessment: Based on results from comparative analyses of data from field trials located at representative sites and environments in Canada in 1995-1997, it is concluded that oilseed rape T45 is agronomically and phenotypically equivalent to the conventional counterpart and commercial available reference varieties, with the exception of maturity and the herbicide tolerance conferred by the PAT protein. The field evaluations support a conclusion of no phenotypic changes indicative of increased plant weed/pest potential of event T45 compared to conventional oilseed rape. Furthermore, the results demonstrate that in-crop applications of glufosinate herbicide do not alter the phenotypic and agronomic characteristics of event T45 compared to conventional oilseed rape. Environmental risk: According to the applicant, the event T45 has been phased out, and stocks of all oilseed rape T45 lines have been recalled from distribution and destroyed since 2005. However, since future cultivation and import of oilseed rape T45 into the EU/EEA area cannot be entirely ruled out, the environmental risk assessment consider exposure of viable seeds of T45 through accidental spillage into the environment during transportation, storage, handling, processing and use of derived products. Oilseed rape is mainly a self-pollinating species, but has entomophilous flowers capable of both self- and cross-pollinating. Normally the level of outcrossing is about 30%, but outcrossing frequencies up to 55% are reported. Several plant species related to oilseed rape that are either cultivated, occurs as weeds of cultivated and disturbed lands, or grow outside cultivation areas to which gene introgression from oilseed rape could be of concern. These are found both in the Brassica species complex and in related genera. A series of controlled crosses between oilseed rape and related taxa have been reported in the scientific literature. Because of a mismatch in the chromosome numbers most hybrids have a severely reduced fertility. Exceptions are hybrids obtained from crosses between oilseed rape and wild turnip (B. rapa ssp. campestris) and to a lesser extent, mustard greens (B. juncea), where spontaneously hybridising and transgene introgression under field conditions have been confirmed. Wild turnip is native to Norway and a common weed in arable lowlands. There is no evidence that the herbicide tolerant trait results in enhanced fitness, persistence or invasiveness of oilseed rape T45, or hybridizing wild relatives, compared to conventional oilseed rape varieties, unless the plants are exposed to herbicides with the active substance glufosinate ammonium. Glufosinate ammonium-containing herbicides have been withdrawn from the Norwegian market since 2008, and the substance will be phased out in the EU in 2017 for reasons of reproductive toxicity. Accidental spillage and loss of viable seeds of T45 during transport, storage, handling in the environment and processing into derived products is, however, likely to take place over time, and the establishment of small populations of oilseed rape T45 cannot be excluded. Feral oilseed rape T45 arising from spilled seed could theoretically pollinate conventional crop plants if the escaped populations are immediately adja

Preliminary Environmental Risk Assessment of Insect Resistant Genetically Modified Maize MON 89034 for Cultivation (EFSA/GMO/BE/2011/90)

The environmental risk assessment of the insect resistant genetically modified maize MON 89034 (Reference EFSA/GMO/BE/2011/90) has been performed by the Panel on Genetically Modified Organisms (GMO) of the Norwegian Scientific Committee for Food Safety (VKM). VKM has been requested by the Norwegian Directorate for Nature Management and the Norwegian Food Safety Authority to issue a preliminary scientific opinion on the safety of the genetically modified maize MON 89034 (Unique identifier MON-89Ø34-3) for cultivation, and submit relevant scientific comments or questions to EFSA on the application EFSA/GMOBE/2011/90. The current submission is intended to complement application EFSA-GMO-NL-2007-37, which was approved by Commission Decision 2009/813/EC of 30 October 2009, authorising the placing on the market of products containing, consisting of, or produced from genetically modified maize MON 89034 (scope import, processing, food and feed). Maize MON89034 has previously been assessed by the VKM GMO Panel in connection with EFSA´s public hearing of the application EFSA/GMO/NL/2007/37 (VKM 2008a). Preliminary health- and environmental risk assessments of several stacked events, with MON 89034 as one of the parental lines, have also been performed by the VKM GMO Panel (VKM 2009a, b, c; VKM 2010a,b). The environmental risk assessment of the maize MON 89034 is based on information provided by the applicant in the application EFSA/GMO/BE/2011/90, and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated MON 89034 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2006, 2011a), the environmental risk assessment of GM plants (EFSA 2010), the selection of comparators for the risk assessment of GM plants (EFSA 2011b), and for the post-market environmental monitoring of GM plants (EFSA 2006, 2011c). The scientific risk assessment of maize MON 89034 include molecular characterisation of the inserted DNA and expression of target proteins, comparative assessment of agronomic and phenotypic characteristics, unintended effects on plant fitness, potential for gene transfer, interactions between the GM plant and target and non-target organisms, effects on biogeochemical processes and evaluations of the post-market environmental plan. In line with its mandate, VKM emphasised that assessments of sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act, shall not be carried out by the Panel on Genetically Modified Organisms. The genetically modified maize MON 89034 was developed to provide protection against certain lepidopteran target pest, including European corn borer (Ostrinia nubilalis) and Mediterranean corn borer (Sesamia nonagrioides). Protection is achieved through expression in the plant of two insecticidal Cry proteins, Cry1A.105 and Cry2Ab2, derived from Bacillus thuringiensis, a common soil bacterium. Cry1A.105, encoded by the cry1A.105 gene, is a chimeric protein made up of different functional domains derived from three wild-type Cry proteins from B. thuringiensis subspecies kurstaki and aizawai. The Cry2Ab2 protein is encoded by the cry2Ab2 gene derived from B. thuringiensis subspecies kurstaki. Molecular characterization: Appropriate analysis of the integration site, including flanking sequence and bioinformatics analysis, has been performed to characterise the transformation event MON 89034. The results of the segregation analysis are consistent with a single site of insertion for the cry1A.105 and cry2Ab2 gene expression cassettes and confirm the results of the molecular characterisation. Molecular analysis of both self-pollinated and cross-fertilised lines, representing a total of seven different generations, indicates that the inserted DNA is stably transformed and inherited from one generation to the next. No genes that encode resistance to antibiotics are present in the genome of MON 89034 maize. The molecular characterisation confirmed the absence of both the aad and nptII genes, which were used in the cloning and transformation process. Event MON 89034 and the physical, chemical and functional characteristics of the proteins have previously been evaluated by The VKM Panel on Genetically Modified Organisms, and considered satisfactory (VKM 2008a). Comparative assessment: The field trials for comparative assessment of agronomic and phenotypic characteristics of maize MON 89034 in the USA (2004-2005) and Europe (2007), have been performed in accordance with the EFSAs guidelines for risk assessment of genetically modified plants and derived food and feed (EFSA 2010, 2011a). Based on results from the comparative analyses, it is concluded that maize MON 89034 is agronomically and phenotypically equivalent to the conventional counterpart and commercial available reference varieties, with the exception of the lepidopteran-protection trait. The field evaluations support a conclusion of no phenotypic changes indicative of increased plant weed/pest potential of MON 89034 compared to conventional maize. Evaluations of ecological interactions between maize MON 89034 and the biotic and abiotic environment indicate no unintended effects of the introduced trait on agronomic and phenotypic characteristics. Environmental risk: There are no reports of the target Lepidopteran species attaining pest status on maize in Norway. Since there are no Bt-based insecticides approved for use in Norway, and lepidopteran pests have not been registered in maize, issues related to resistance evolution in target pests are not relevant at present for Norwegian agriculture. Published scientific studies show no or negligible adverse effects of Cry1A.105 and Cry2Ab2 proteins on non-target arthropods that live on or in the vicinity of maize plants. Cultivation of maize MON 89034 is not considered to represent a threat to the prevalence of red-listed species in Norway. Few studies have been published examining potential effects of Cry1A.105 and Cry2Ab toxin on ecosystems in soil, mineralization, nutrient turnover and soil communities. Some field studies have indicated that root exudates and decaying plant material containing Cry proteins may affect population size and activity of rhizosphere organisms (soil protozoa and microorganisms). However, data are only available from short term experiments and predictions of potential long term effects are difficult to deduce. Most studies conclude that effects on soil microorganisms and microbial communities are transient and minor compared to effects caused by agronomic and environmental factors. Few studies have assessed the impact of Cry proteins on non-target aquatic arthropods and the fate of these proteins in senescent and decaying maize detritus in aquatic environments. Further studies with better experimental design are needed for the assessment of the potential effects of Bt crops on aquatic organisms. However, exposure of non-target organisms to Cry proteins in aquatic ecosystems is likely to be very low, and potential exposure of Bt toxins to non-target organisms in stream ecosystems in Norway is considered to be negligible. Maize is the only representative of the genus Zea in Europe, and there are no cross-compatible wild or weedy relatives outside cultivation with which maize can hybridise and form backcross progeny. Vertical gene transfer in maize therefore depends on cross-pollination with other conventional or organic maize varieties. In addition, unintended admixture of genetically modified material in seeds represents a possible way for gene flow between different crop cultivations. The risk of pollen flow from maize volunteers is negligible under Norwegian growing conditions. In addition to the data presented by the applicant, the VKM GMO Panel is not aware of any scientific report of increased establishment and spread of maize MON 89034 and any change in survival (including over-wintering), persistence and invasiveness capacity. Because the general characteristics of maize MON 89034 are unchanged, insect resistance are not likely to provide a selective advantage outside cultivation in Norway. Since MON 89034 has no altered agronomic and phenotypic characteristics, except for the specific target pest resistance, the VKM GMO Panel is of the opinion that the likelihood of unintended environmental effects due to the establishment and survival of maize MON 89034 will be no different to that of conventional maize varieties in Norway The environmental risk assessment will be completed and finalized by the VKM Panel on Genetically Modified Organisms when requested additional information from the applicant is available.

Risk Assessment of Insect-resistant and Herbicide Tolerant Genetically Modified Maize Bt11 for Cultivation, Import, Processing, Food and Feed Uses under Directive 2001/18/EC and Regulation (EC) Opinion

The Norwegian Environment Agency (NEA) and the Norwegian Food Safety Authority (NFSA) requested the Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) for an opinion of potential risks to biodiversity and agriculture in Norway associated with import of seeds for sowing and cultivation of insect-resistant and herbicide tolerant genetically modified maize Bt11 under Directive 2001/18/EC (Notification C/F/96.05.10). The notification is still pending for authorisation in the European Union. VKM is also requested to assess the applicant´s post-market environmental monitoring plan, and the management measures suggested in the draft implementing decision of the European Commission. As the scope of the notification does not cover food and feed uses of maize Bt11, VKM was not asked for a health risk assessment of maize Bt11. However, VKM has decided to update a previous safety evaluation of the food and feed uses of maize Bt11 and derived products (VKM, 2014). VKM appointed a working group consisting of members from the Panel on Genetically Modified Organisms, the Panel on Alien Organisms and trade in Endangered Species (CITES) and the VKM staff to answer the requests. The Panel on Genetically Modified Organisms assessed and approved the final report. The genetically modified maize Bt11 was developed to provide protection against certain lepidopteran target pests, such as the European corn borer (ECB, Ostrinia nubilalis), and some species belonging to the genus Sesamia . The insect resistence is achieved by the expression of a truncated form of a Cry1Ab protein encoded by a modified cry1Ab gene derived from the soil microorganism Bacillus thuringiensis subsp kurstaki HD-1. Maize Bt11 also expresses the phosphinothricin - N - ace tyltransferase (pat) gene, derived from the soil microorganism Streptomyces viridochromogenes strain Tu494, which encodes the enzyme: phosphinothricin acetyl transferase (PAT). PAT protein confers tolerance to the herbicidal active substance glufosinate-ammonium. The PAT protein expressed in Bt11 was used as a selectable marker to facilitate the selection process of transformed plant cells and is not intended for weed management purposes. Since the scope of the notification C/F/96.05.10 does not cover the use of glufosinate-ammonium-containing herbicides on maize Bt11, potential effects due to the use of such herbicides on maize Bt11 are not considered by VKM. In delivering its scientific opinion, VKM considered relevant peer-reviewed scientific publications and information provided by the applicant in the notification C/F/96.05.10, the renewal application EFSA/GMO/RX/Bt11, and scientific opinions and comments from EFSA and other EU-member states. VKM has evaluated maize Bt11 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2011a), the environmental risk assessment of GM plants (EFSA 2010a), selection of comparators for the risk assessment of GM plants (EFSA, 2011b) and for the post-market environmental monitoring of GM plants (EFSA, 2011c). The scientific risk assessment of maize Bt11 includes molecular characterisation of the inserted DNA and expression of novel proteins, comparative assessment of agronomic and phenotypic characteristics, nutritional assessments, toxicology and allergenicity. An evaluation of unintended effects on plant fitness, potential for gene transfer, interactions between the GM plant and target and non-target organisms, effects on biogeochemical processes, the post-market environmental monitoring plan and coexistence measures at the farm level has also been undertaken. It is emphasised that the VKM mandate does not include assessments of contribution to sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act. These considerations are therefore not part of the risk assessment provided by the VKM. Molecular Characterization: Appropriate analyses of the integration site, inserted DNA sequence, flanking regions, and bioinformatics have been performed. The molecular characterisation reported by the applicant shows that the DNA-fragment containing the cry1Ab and pat genes, is integrated as a single copy at a single locus in the nuclear genome of maize Bt11 and that it is stably inherited as a dominant trait. VKM considers the molecular characterisation of maize Bt11 satisfactory. Comparative Assessment: Comparative analyses of data from field trials located at representative sites and environments in North America and Europe indicates that maize Bt11 is compositionally equivalent to its conventional counterpart, with the exception of the herbicide tolerance and insect resistance traits, conferred by the expression of the PAT and Cry1Ab proteins. However, data on the amino acid tryptophan, is only given in one out of six studies. Based on current knowledge, VKM concludes that maize Bt11 is compositionally equivalent to conventional maize. The data provided by the applicant are not sufficient to show that Bt11 maize is phenotypically and agronomically equivalent to conventional near-isogenic maize lines. The agronomic assessment data are provided from one growing season in the North America and one growing season in France. This is not considered to be sufficient for representative testing of agricultural environments. Food and Feed Risk Assessment: Whole food feeding studies have not indicated any adverse health effects of maize Bt11. These studies further support that maize Bt11 is nutritionally equivalent to conventional maize. The Cry1Ab and PAT proteins do not show sequence resemblance to other known toxins or IgE allergens, nor have they been reported to cause IgE mediated allergic reactions. Some studies have however indicated a potential role of Cry-proteins as adjuvants in allergic reactions. Based on current knowledge, the VKM concludes that maize Bt11 is nutritionally equivalent to conventional maize varieties. It is unlikely that the Cry1Ab and PAT proteins will introduce a toxic or allergenic potential in food or feed based on maize Bt11 compared to conventional maize. Environmental Risk Assessment: Maize is the only representative of the genus Zea in Europe, and there are no cross-compatible wild or weedy relatives outside cultivated maize with which maize can hybridise and form backcross progeny. Vertical gene transfer in maize therefore depends on cross-pollination with other conventional or organic maize varieties. In addition, unintended admixture of genetically modified material in seeds represents a possible way for gene flow between different crop cultivations. The risk of pollen flow from maize volunteers is negligible under Norwegian growing conditions. Since maize Bt11 has no altered agronomic and phenotypic characteristics, except for the specific target insect resistance and herbicide tolerance, the likelihood of unintended environmental effects as a consequence of spread of genes from maize Bt11 is considered to be extremely low. There are no reports of the target lepidopteran species attaining pest status on maize in Norway. Since there are no Bt-based insecticides approved for use in Norway, and lepidopteran pests have not been registered in maize, issues related to resistance evolution in target pests are not relevant at present for Norwegian agriculture. Published scientific studies showed that the likelihood of negative effects of Cry1Ab protein on non-target arthropods that live on or in the vicinity of maize plants is low. In Norway, the maize cultivation is marginal. The total crop area of forage maize is estimated to 2000-2800 decares, equivalent to less than 0.1% of the areas with cereal crops. The area of individual fields is limited by the topography such that the quantity of maize pollen produced under flowering is also limited. The potential exposure of Cry1Ab-containing maize pollen on non-target lepidopteran species in Norway is therefore negligible. Cultivation of maize Bt11 is not considered to represent a threat to the prevalence of red-listed species in Norway. Exposure of nontarget organisms to Cry proteins in aquatic ecosystems is likely to be very low, and potential exposure of Cry proteins to non-target organisms in aquatic ecosystems in Norway is considered to be negligible. VKM concludes that, although the data on the fate of the Cry1Ab protein and its potential interactions in soil are limited, the relevant scientific publications analysing the Cry1Ab protein, together with the relatively broad knowledge about the environmental fate of other Cry1 proteins, do not indicate significant direct effects on the soil environment. Despite limited number of studies, most studies conclude that effects on soil microorganisms and microbial communities are transient and minor compared to effects caused by agronomic and environmental factors. However, data are only available from short-term experiments and predictions of potential long-term effects are difficult to deduce. Coexistence: VKM concludes that separation distances of 200 meters most likely will ensure coexistence between genetically modified maize and conventional and organic maize varieties in Norway. Overall Conclusion: Based on current knowledge, VKM concludes that maize Bt11 is nutritionally equivalent t

Final Health and Environmental Risk Assessment of Genetically Modified Maize MON 89034 x NK 603

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Scientific Committee for Food Safety (VKM) has been requested by the Norwegian Environment Agency (former Norwegian Directorate for Nature Management) and the Norwegian Food Safety Authority (NFSA) to conduct final food/feed and environmental risk assessments for all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC. The request covers scope(s) relevant to the Gene Technology Act. The request does not cover GMOs that VKM already has conducted its final risk assessments on. However, the Agency and NFSA requests VKM to consider whether updates or other changes to earlier submitted assessments are necessary. The insect-resistant and glyphosate-tolerant genetically modified maize MON 89034 x NK 603 from Monsanto (Unique Identifier MON-89Ø34-3 × MON-ØØ6Ø3-6) was approved under Regulation (EC) No 1829/2003 in the EU for food and feed uses, import and processing on 28 July 2010 (Commission Decision 2010/420/EC). Genetically modified maize MON 890314 x NK 603 has previously been risk assessed by the VKM Panel on Genetically Modified Organisms (GMO), commissioned by the Norwegian Food Safety Authority and the Norwegian Environment Agency related and to the EFSA public hearing of the applications EFSA/GMO/NL/2007/38 and EFSA/GMO/NL/2009/72 in 2007 and 2009/2010 (VKM 2008a, VKM 2010a). In addition, the parental lines MON 89034 and NK 603 have been evaluated by the VKM GMO Panel as single events and as a component of several stacked GM maize events (VKM 2005a,b,c,d,e, VKM 2007a,b, VKM 2008b,c,d, VKM 2009a,b, VKM 2010 a,b, VKM 2011, VKM 2012a,b, VKM 2013 a,b, VKM 2014). The food/feed and environmental risk assessment of the maize MON 89034 x NK 603 is based on information provided by the applicant in the applications EFSA/GMO/NL/2007/38 EFSA/GMO/NL/2009/72 and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated MON 89034 x NK 603 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2011a), the environmental risk assessment of GM plants (EFSA 2010a), selection of comparators for the risk assessment of GM plants (EFSA 2011b) and for the post-market environmental monitoring of GM plants (EFSA 2011c). The scientific risk assessment of maize MON 89034 x NK 603 include molecular characterisation of the inserted DNA and expression of novel proteins, comparative assessment of agronomic and phenotypic characteristics, nutritional assessments, toxicology and allergenicity, unintended effects on plant fitness, potential for gene transfer, effects on biogeochemical processes and interactions between the GM plant and target and non-target organisms. It is emphasized that the VKM mandate does not include assessments of contribution to sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act. These considerations are therefore not part of the risk assessment provided by the VKM Panel on Genetically Modified Organisms. Likewise, the VKM mandate does not include evaluations of herbicide residues in food and feed from genetically modified plants. The hybrid maize MON 89034 x NK 603 has been produced by conventional crosses between inbred lines containing MON 89034 and NK 603 events to combine resistance to certain lepidopteran pests and to confer tolerance towards glyphosate-containing herbicides. Maize MON 89034 was developed to provide protection against specific lepidopteran target pest, including Ostrinia nubilalis, S podoptera spp. and Agrotis ipsilon. Protection is achieved through expression in the plant of two insecticidal Cry proteins, Cry1A.105 and Cry2Ab2, derived from Baci llus thuringiensis subsp. a izawai and kurstaki. Maize NK 603 has been developed to provide tolerance to glyphosate by the introduction, of a gene coding for 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 (CP4 EPSPS). Molecular Characterisation: Southern and PCR analyses indicate that the recombinant inserts in the single maize events MON 89034 and NK 603 are retained in maize stack MON 89034 x NK603. Genetic stability of the inserts has previously been demonstrated in the parental lines MON 89034and NK603. The level of Cry1A.105, Cry2Ab2 and CP4 EPSPS proteins in grain and forage from the stacked event are comparable to the levels in the corresponding single events. Phenotypic analyses also indicate stability of the insect resistance and herbicide tolerance traits of the stacked event. Based on current knowledge and the previous assessments of the parental maize events, the VKM GMO Panel considers the molecular characterisation of maize MON 89034 x NK 603 satisfactory. 6 VKM Report 2016: 17. Comparative Assessment: The applicant has performed comparative analyses of data from field trials located at representative sites and environments in Argentina in 2004/2005 and Europe in 2007. With the exception of small intermittent variations and the insect resistance and herbicide tolerance conferred by the Cry1A.105, Cry2Ab2 and CP4 EPSPS proteins, the results showed no biologically relevant differences between maize stack MON 89034 x NK 603 and conventional control. Based on the assessment of available data, the VKM GMO Panel concludes that maize MON 89034 x NK 603 is compositionally, agronomical and phenotypically equivalent to its conventional counterpart, except for the new proteins. Food/feed Safety Assessment: A whole food feeding study on broilers has not indicated any adverse health effects of maize MON 89034 x NK 603, and shows that it is nutritionally equivalent to conventional maize varieties. The Cry1A.105, Cry2Ab2, and CP4 EPSPS proteins do not show sequence resemblance to other known toxins or IgE allergens, nor have they been reported to cause IgE mediated allergic reactions. However, some studies have indicated a potential role of Cry-proteins as adjuvants in allergic reactions. Based on current knowledge, the VKM GMO Panel concludes that maize MON 89034 x NK 603 is nutritionally equivalent to conventional maize varieties. It is unlikely that the Cry1A.105, Cry2Ab2, and CP4 EPSPS proteins will cause toxic or IgE-mediated allergic reactions to food or feed based on maize MON 89034 x NK 603 compared to conventional maize. Environmental Risk: Considering the intended uses of maize MON 89034 x NK603, excluding cultivation, the environmental risk assessment is concerned with accidental release into the environment of viable grains during transportation and processing, and indirect exposure, mainly through manure and faeces from animals fed grains from maize MON 89034 x NK603. Maize MON 89034 x NK 603 has no altered survival, multiplication or dissemination characteristics, and there are no indications of an increased likelihood of spread and establishment of feral maize plants in the case of accidental release into the environment of seeds from maize MON 89034 x NK603. Maize is the only representative of the genus Zea in Europe, and there are no cross-compatible wild or weedy relatives outside cultivation. The VKM GMO Panel considers the risk of gene flow from occasional feral GM maize plants to conventional maize varieties to be negligible in Norway. Considering the intended use as food and feed, interactions with the biotic and abiotic environment are not considered by the GMO Panel to be an issue. 7 VKM Report 2016: 17. Overall Conclusion: Based on current knowledge, the VKM GMO Panel concludes that maize MON 89034 x NK 603 is compositionally, nutritionally, agronomically and phenotypically equivalent to its conventional counterpart except for the new proteins. It is unlikely that the Cry1A.105, Cry2Ab2 and CP4 EPSPS proteins will cause an increased risk of toxic or IgE-mediated allergic reactions to food or feed based on maize MON 89034 x NK 603 compared to conventional maize varieties. The VKM GMO Panel concludes that maize MON 89034 x NK603, based on current knowledge, is comparable to conventional maize varieties concerning environmental risk in Norway with the intended usage.

Final Health- and Environmental Risk Assessment of Genetically Modified Maize MON 89034 x MON 88017

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Scientific Committee for Food Safety (VKM) has been requested by the Norwegian Environment Agency and the Norwegian Food Safety Authority (NFSA) to conduct final food/feed and environmental risk assessments for all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC. The request covers scope(s) relevant to the Gene Technology Act. The request does not cover GMOs that VKM already has conducted its final risk assessments on. However, the Agency and NFSA requests VKM to consider whether updates or other changes to earlier submitted assessments are necessary. The insect-resistant and glyphosate-tolerant genetically modified maize MON 89034 x MON 88017 from Monsanto (Unique Identifier MON-89Ø34-3 × MON-88Ø17-3) was approved under Regulation (EC) No 1829/2003 in the EU for food and feed uses, import and processing on 17th of June 2011 (Commission Decision 2011/366/EC). Genetically modified maize MON 890314 x MON 88017 has previously been risk assessed by the VKM Panel on Genetically Modified Organisms (GMO), commissioned by the Norwegian Food Safety Authority and the Norwegian Environment Agency related and to the EFSA public hearing of the applications EFSA/GMO/NL/2007/39 and EFSA/GMO/BE/2009/71 in 2007 and 2009/2010 (VKM 2008a, VKM 2010a). In addition, the parental lines MON 89034 and MON 88017 have been evaluated by the VKM GMO Panel as single events and as a component of several stacked GM maize events (VKM 2007a,b, VKM 2008b, VKM 2009a,b,c, VKM 2010b,c, VKM 2012, VKM 2013, VKM 2014). The food/feed and environmental risk assessment of the maize MON 89034 x MON 88017 is based on information provided by the applicant in the applications EFSA/GMO/NL/2007/39 EFSA/GMO/BE/2009/71 and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature when relevant. The VKM GMO Panel has evaluated MON 89034 x MON 88017 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2011a), the environmental risk assessment of GM plants (EFSA 2010), selection of comparators for the risk assessment of GM plants (EFSA 2011b) and for the post-market environmental monitoring of GM plants (EFSA 2011c). The scientific risk assessment of maize MON 89034 x MON 88017 include molecular characterisation of the inserted DNA and expression of novel proteins, comparative assessment of agronomic and phenotypic characteristics, nutritional assessments, toxicology and allergenicity, unintended effects on plant fitness, potential for gene transfer, effects on biogeochemical processes and interactions between the GM plant and target and non-target organisms. It is emphasised that the VKM mandate does not include assessments of contribution to sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act. These considerations are therefore not part of the risk assessment provided by the VKM Panel on Genetically Modified Organisms. Likewise, the VKM mandate does not include evaluations of herbicide residues in food and feed from genetically modified plants.The hybrid maize MON 89034 x MON 88017 has been produced by conventional crosses between inbred lines containing MON 89034 and MON 88017 events to combine resistance to certain coleopteran and lepidopteran pests, and to confer tolerance towards glyphosate-containing herbicides. Maize MON 89034 was developed to provide protection against specific lepidopteran target pest, including Ostrinia nubilalis , S podoptera spp. and Agrotis ipsilon. Protection is achieved through expression in the plant of two insecticidal Cry proteins, Cry1A.105 and Cry2Ab2, derived from Bacillus thuringiensis subsp. a izawai and kurstaki. Maize MON 88017 was developed to express a modified Cry3Bb1 insecticidal protein, derived from B. thuringiensis subsp. kumamotoensis , which confers protection against coleopteran target pests belonging to the genus Diabrotica such as Western corn rootworm ( D . virgifera virgifera ). MON 88017 is also developed to provide tolerance to the herbicidal active substance glyphosate by the introduction of a gene coding for the enzyme 5enolpyruvylshikimate-3-phosphate synthase (EPSPS), from Agrobacterium tumefaciens strain CP4 (CP4 EPSPS). Molecular Characterisation: Southern and PCR analyses indicate that the recombinant inserts in the single maize events MON 89034 and MON 88017 are retained in the stacked event MON 89034 x MON 88017. Genetic stability of the inserts has previously been demonstrated in the single events. The levels of Cry1A.105, Cry2Ab2, CP4 EPSPS and Cry3Bb1 proteins in grain and forage from the stacked event are comparable to the levels in the corresponding single events. Phenotypic analyses also indicate stability of the insect resistance and herbicide tolerance traits of the stacked event. Based on current knowledge and the previous assessments of the parental maize events, the VKM GMO Panel considers the molecular characterisation of maize MON 89034 x MON 88017 satisfactory. Comparative Assessment: Comparative analyses of maize MON 89034 x MON 88017 and its conventional counterpart have been performed by the applicant during field trials located at representative sites and environments in USA during 2004, and in Europe in 2007. Several different conventional maize varieties were included in the field trials and used as references. With the exception of small variations, and the insect resistance and herbicide tolerance conferred by the Cry3Bb1, Cry1A105, Cry2Ab2, and CP4 EPSPS proteins, the results from these studies showed no biologically relevant differences between the maize stack MON 89034 x MON 88017 and its conventional counterpart. Based on the assessment of available data, the VKM GMO Panel concludes that maize MON 89034 x MON 88017 is compositionally, agronomically and phenotypically equivalent to its conventional counterpart, except for the new proteins. Food and Feed Safety Assessment: A whole food feeding study performed on broilers indicates no adverse health effects of maize MON 89034 x MON 88017, and shows that it is nutritionally equivalent to conventional maize varieties. The Cry1A.105, Cry2Ab2, Cry3Bb1 and CP4 EPSPS proteins do not show relevant sequence resemblance to other known toxins or IgE-allergens, nor have they been reported to cause IgE-mediated allergic reactions. However, some studies have indicated a potential role of Cry-proteins as adjuvants in allergic reactions. Based on current knowledge, the VKM GMO Panel concludes that maize MON 89034 x MON 88017 is nutritionally equivalent to conventional maize varieties. It is unlikely that the Cry1A.105, Cry2Ab2, Cry3Bb1 and CP4 EPSPS proteins will cause toxic or IgE-mediated allergic reactions to food or feed derived from maize MON 89034 x MON 88017 compared to conventional maize. Environmental Risk: Considering the intended uses of maize MON 89034 x MON 88017, excluding cultivation, the environmental risk assessment is concerned with accidental release into the environment of viable grains during transportation and processing, and indirect exposure, mainly through manure and faeces from animals fed grains from maize MON 89034 x MON 88017. Maize MON 89034 x MON 88017 has no altered survival, multiplication or dissemination characteristics, and there are no indications of an increased likelihood of spread and establishment of feral maize plants in the case of accidental release into the environment of seeds from maize MON 89034 x MON 88017. Maize is the only representative of the genus Zea in Europe, and there are no cross-compatible wild or weedy relatives outside cultivation. The VKM GMO Panel considers the risk of gene flow from occasional feral GM maize plants to conventional maize varieties to be negligible in Norway. Considering the intended use as food and feed, interactions with the biotic and abiotic environment are not considered by the GMO Panel to be an issue. Overall Conclusion: Based on current knowledge, the VKM GMO Panel concludes that maize MON 89034 x MON 88017 is compositionally, nutritionally, agronomically and phenotypically equivalent to its conventional counterpart except for the new proteins. It is unlikely that the Cry1A.105, Cry2Ab2, CryBb1 and CP4 EPSPS proteins will cause an increased risk of toxic or IgE-mediated allergic reactions to food or feed based on maize MON 89034 x MON 88017 compared to conventional maize varieties. The VKM GMO Panel concludes that maize MON 89034 x MON 88017, based on current knowledge, is comparable to conventional maize varieties concerning environmental risk in Norway with the intended usage.

Final Health- and Environmental Risk Assessment of Genetically Modified Maize MON 88017 x MON 810

In preparation for a legal implementation of EU-regulation 1829/2003, the Norwegian Scientific Committee for Food Safety (VKM) has been requested by the Norwegian Environment Agency and the Norwegian Food Safety Authority (NFSA) to conduct final food/feed and environmental risk assessments for all genetically modified organisms (GMOs) and products containing or consisting of GMOs that are authorized in the European Union under Directive 2001/18/EC or Regulation 1829/2003/EC. The request covers scope(s) relevant to the Gene Technology Act. The request does not cover GMOs that VKM already has conducted its final risk assessments on. However, the Agency and NFSA requests VKM to consider whether updates or other changes to earlier submitted assessments are necessary. The insect-resistant and glyphosate-tolerant genetically modified maize MON 88017 x MON 810 from Monsanto (Unique Identifier DAS-MON 88017-3 x MON-ØØ81Ø-6) was approved under Regulation (EC) No 1829/2003 in the EU for food and feed uses, import and processing on 28th of July 2010 (Commission Decision 2010/429/EC). Genetically modified maize MON 88017 x MON 810 has previously been risk assessed by the VKM Panel on Genetically Modified Organisms (GMO), commissioned by the Norwegian Food Safety Authority related to the EFSA public hearing of the application in 2007 (VKM 2007a). In addition, MON 88017 and MON 810 has been evaluated by the VKM GMO Panel as single events and as a component of several stacked GM maize events and Regulation (EC) 1829/2003 and Directive 2001/18/EC (VKM 2005a,b,c, VKM 2007b,c,d, VKM 2008, VKM 2009, VKM 2010 a,b,c, VKM 2012, VKM 2013, VKM 2016). The food/feed and environmental risk assessment of the maize MON 88017 x MON 810 is based on information provided by the applicant in the application EFSA/GMO/CZ/2006/33 and scientific comments from EFSA and other member states made available on the EFSA website GMO Extranet. The risk assessment also considered other peer-reviewed scientific literature as relevant. The VKM GMO Panel has evaluated MON 88017 x MON 810 with reference to its intended uses in the European Economic Area (EEA), and according to the principles described in the Norwegian Food Act, the Norwegian Gene Technology Act and regulations relating to impact assessment pursuant to the Gene Technology Act, Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms, and Regulation (EC) No 1829/2003 on genetically modified food and feed. The Norwegian Scientific Committee for Food Safety has also decided to take account of the appropriate principles described in the EFSA guidelines for the risk assessment of GM plants and derived food and feed (EFSA 2011a), the environmental risk assessment of GM plants (EFSA 2010), selection of comparators for the risk assessment of GM plants (EFSA 2011b) and for the post-market environmental monitoring of GM plants (EFSA 2011c). The scientific risk assessment of maize MON 88017 x MON 810 include molecular characterisation of the inserted DNA and expression of novel proteins, comparative assessment of agronomic and phenotypic characteristics, nutritional assessments, toxicology and allergenicity, unintended effects on plant fitness, potential for gene transfer, effects on biogeochemical processes and interactions between the GM plant and target and non-target organisms. It is emphasized that the VKM mandate does not include assessments of contribution to sustainable development, societal utility and ethical considerations, according to the Norwegian Gene Technology Act and Regulations relating to impact assessment pursuant to the Gene Technology Act. These considerations are therefore not part of the risk assessment provided by the VKM Panel on Genetically Modified Organisms. Likewise, the VKM mandate does not include evaluations of herbicide residues in food and feed from genetically modified plants. The hybrid maize MON 88017 x MON 810 was produced by conventional crosses between inbred lines containing MON 88017 and MON 810 events to combine resistance to certain coleopteran and lepidopteran pests, and to confer tolerance towards glyphosate-containing herbicides. Maize MON 88017 was developed to express a modified Cry3Bb1 insecticidal protein, derived from Bacillus thuringiensis subsp. kumamotoensis , which confers protection against coleopteran target pests belonging to the genus Diabrotica such as Western corn rootworm ( Diabrotica virgifera virgifera ). MON 88017 is also developed to provide tolerance to the herbicidal active substance glyphosate by the introduction of a gene coding for the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), from Agrobacteri um tumefaciens strain CP4 (CP4 EPSPS). Maize MON 810 expresses the Cry1Ab insecticidal protein, derived from Bacillus thuringiensis subsp. k u rstaki, which confers protection against lepidopteran pests such as Ostrinia nubilaris and species belonging to the genus Sesamia. Molecular characterisation Southern and PCR analyses indicate that the recombinant inserts in the single maize events MON 88017 and MON 810 are retained in the stacked event MON 88017 x MON 810. Genetic stability of the inserts has previously been demonstrated in the single events. The levels of CP4 EPSPS, Cry3Bb1 and Cry1Ab proteins in grain and forage from the stacked event are comparable to the levels in the corresponding single events. Phenotypic analyses also indicate stability of the insect resistance and herbicide tolerance traits of the stacked event. Based on current knowledge and the previous assessments of the parental maize events, the VKM GMO Panel considers the molecular characterisation of maize MON 88017 x MON 810 satisfactory. Comparative assessment The applicant has performed comparative analyses of data from field trials located at representative sites and environments in USA during the 2002 growing season. With the exception of small intermittent variations and the insect resistance and herbicide tolerance conferred by the CP4 EPSPS, Cry3Bb1 and Cry1Ab proteins, the results showed no biologically relevant differences between maize stack MON 88017 x MON 810 and its conventional counterpart. Based on the assessment of available data, the VKM GMO Panel concludes that maize MON 88017 x MON 810 is compositionally, agronomically and phenotypically equivalent to its conventional counterpart, except for the new proteins. Food and feed safety assessment A whole food feeding study on broilers indicates no adverse health effects of maize MON 88017 x MON 810, and shows that it is nutritionally equivalent to conventional maize varieties. The Cry3Bb1, Cry1Ab and CP4 EPSPS proteins do not show relevant sequence resemblance to other known toxins or IgE-allergens, nor have they been reported to cause IgE-mediated allergic reactions. However, some studies have indicated a potential role of Cry-proteins as adjuvants in allergic reactions. Based on current knowledge, the VKM GMO Panel concludes that maize MON 88017 x MON 810 is nutritionally equivalent to conventional maize varieties. It is unlikely that the Cry3Bb1, Cry1Ab and CP4 EPSPS proteins will cause toxic or IgE-mediated allergic reactions to food or feed based on maize MON 88017 x MON 810 compared to conventional maize. Environmental risk assessment Considering the intended uses of maize MON 88017 x MON 810, excluding cultivation, the environmental risk assessment is concerned with accidental release into the environment of viable grains during transportation and processing, and indirect exposure, mainly through manure and faeces from animals fed grains from maize MON 88017 x MON 810. Maize MON 88017 x MON 810 has no altered survival, multiplication or dissemination characteristics, and there are no indications of an increased likelihood of spread and establishment of feral maize plants in the case of accidental release into the environment of seeds from maize MON 88017 x MON 810. Maize is the only representative of the genus Zea in Europe, and there are no cross-compatible wild or weedy relatives outside cultivation. The VKM GMO Panel considers the risk of gene flow from occasional feral GM maize plants to conventional maize varieties to be negligible in Norway. Considering the intended use as food and feed, interactions with the biotic and abiotic environment are not considered by the GMO Panel to be an issue. Overall conclusion Based on current knowledge, the VKM GMO Panel concludes that maize MON 88017 x MON 810 is compositionally, nutritionally, agronomically and phenotypically equivalent to its conventional counterpart except for the new proteins. It is unlikely that the Cry3Bb1, Cry1Ab and CP4 EPSPS proteins will cause an increased risk of toxic or IgE-mediated allergic reactions to food or feed based on maize MON 88017 x MON 810 compared to conventional maize varieties. The VKM GMO Panel concludes that maize MON 88017 x MON 810, based on current knowledge, is comparable to conventional maize varieties concerning environmental risk in Norway with the intended usage.