Report on the In-house Validation of a DNA Extraction Method from Oilseed rape Grains and Validated Method

In accordance with relevant EU legislation , Pioneer Overseas Corporation provided to the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) a DNA extraction method for oilseed rape grains and the relevant samples (ground oilseed rape grains). In line with its mandate , the EU RL GMFF has conducted an in-house validation of this DNA extraction method. To this end it tested the DNA extraction method on the samples provided and evaluated its performance in terms of DNA yield, integrity and quality. The in-house validation study confirmed that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL , and that it satisfies the provisions of Annex I-2.C.2 to Regulation (EC) No 641/2004. The method is therefore fit for the purpose of producing rapeseed DNA of suitable quantity and quality for subsequent PCR-based analysis. This report is published at http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.htm.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Maize DAS-40278-9 by Real-time PCR - Validation Report and Validated Method

In line with its mandate1 the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), in collaboration with the European Network of GMO Laboratories (ENGL), has validated an event-specific polymerase chain reaction (PCR) method for detecting and quantifying maize event DAS-40278-9 (unique identifier DAS-4Ø278-9). The validation study was conducted according to the EU-RL GMFF validation procedure [http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm] and the internationally accepted guidelines2. In accordance with current EU legislation1, Dow AgroSciences LLC has provided the detection method and the positive and negative control samples (genomic DNA from maize seeds harbouring the DAS-40278-9 event as positive control DNA, genomic DNA from conventional maize seeds as negative control DNA). The EU-RL GMFF prepared the validation samples (calibration samples and blind samples at different GM percentage [DNA/DNA]), organised an international collaborative study and analysed the results. The study confirms that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL in Annex I-2.C.2 to Regulation (EC) No 641/20041 and it fulfils the analytical requirements of Regulation (EU) No 619/2011. This report is published at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm.JRC.I.3-Molecular Biology and Genomic

Report on the single-laboratory validation of a PCR-based Detection Method for Identification of Florigene™ IFD-25958-3 GM Carnation -Validation Report and Validated Method

In the context of the application for marketing submitted by Florigene Pty Ltd for a genetically modified carnation line (C/NL/09/01) IFD-25958-3, the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) has carried out a single-laboratory validation to assess the performance of a polymerase chain reaction (PCR)-based detection method for detecting and identifying the carnation GM line IFD-25958-3. This report describes the results of tests carried out by the EU-RL GMFF on control samples provided by the method developer and according to the detection method described by the applicant. The taxon-specific method correctly detects the endogenous gene target in genomic DNA of a conventional carnation line (negative control) and in the genomic DNA of the GM carnation line; the same method can also detect the GM target DNA in IFD-25958-3 GM line (positive control) in the experimental conditions described in this report. The Limit of Detection (LOD) of the method has been estimated to be at least 50 copies for the taxon-specific gene and at least 100 copies for the GM insert, based on haploid genome copy number.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Soybean Line A5547-127 Using Real-time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the A5547-127 transformation event in soybean DNA (unique identifier ACS-GMØØ6-4). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 ¿on genetically modified food and feed¿ and with Regulation (EC) No 641/2004 of 6 April 2004 ¿on detailed rules for the implementation of Regulation (EC) No 1829/2003¿, Bayer CropScience provided the detection method and the samples (genomic DNA from leaves of plants harbouring the A5547-127 event and from leaves of conventional A5547 soybean plants). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from eight European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is, therefore, considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Commission Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.it/.JRC.DDG.I.4-Molecular biology and genomic

Report on the Single-Laboratory Validation of a DNA Extraction Method from Carnation Leaves - Validation Report and Validated Method

In the context of the application for marketing submitted by Florigene Pty Ltd for a genetically modified carnation line (C/NL/09/01) IFD-25958-3, the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) has carried out a single-laboratory validation to assess the performance of a DNA extraction protocol to extract genomic DNA from carnation plant leaves for subsequent polymerase chain reaction (PCR) based detection methods and its applicability on the samples provided by the applicant. This report describes the results of tests carried out by the EU-RL GMFF on samples provided by the method developer and according to the DNA extraction method described by the applicant. The data reported confirm that the extraction method, applied to samples of carnation leaves, produces DNA of suitable quantity and quality for subsequent PCR-based methods.JRC.I.3 - Molecular Biology and Genomic

Report on the Verification of the Performance of MON 531 and MON 1445 Event-specific Methods on the Cotton Event MON 531 x MON 1445 Using Real-Time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, has carried out an in-house verification study to assess the performance of two quantitative event-specific methods on the cotton event MON 531 x MON 1445 (unique identifier MON-ØØ531-6 x MON-Ø1445-2) which combines the MON 531 and MON 1445 transformation events. The two methods have been previously validated individually on single-trait events, to detect and quantify each event in cotton samples; a validation report for each method is available at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm. This study was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Europe S.A. provided the detection methods and the control samples: whole seeds of MON 531 x MON 1445 cotton (ST5599BR) and whole conventional cotton seeds of ST474. The JRC prepared the in-house verification samples (calibration samples and blind samples at different GM percentages). The results of the in-house verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the validation results on the individual parental events (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). The results of this CRL-GMFF in-house verification studies are made publicly available at http://gmo-crl.jrc.ec.europa.eu/.JRC.DG.I.4 - Molecular biology and genomic

Report on the Verification of the Performance of a Method for the Detection of Event MON71800 in Wheat Using Real-Time PCR

Following the United States Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS) announcement that test results confirmed the finding of unauthorised GM glyphosate-resistant wheat "volunteer" plants harbouring the event MON71800 on a farm in Oregon, the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) was requested to provide as soon as possible a method to test wheat consignments for the presence of this genetically modified organism (GMO) to the National Reference Laboratories (NRLs) for GMOs of the EU Member States. In response, the EU-RL put together a testing strategy, based on readily available screening tests which was published here (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm). Upon request, Monsanto provided in May 2013 the EU-RL with the procedure “Roundup Ready® Wheat MON71800 Event Specific Endpoint TaqMan® PCR with acc Internal Control for Seed Pools of 1:15” that had previously been made available to, and was used by USDA. The EU-RL GMFF tested this protocol on positive control samples consisting of MON71800 crude lysate, also provided by Monsanto. Our results can be summarised as follow: The method is apparently event-specific. Our specificity-tests did not show cross-reactivity on genomic DNA from a wide selection of similar GMO. The sensitivity of the method was found to be in agreement with previous findings of USDA, i.e. the relative limit of detection lies at 0.5% in a background of 301 ng of total wheat genomic DNA. The absolute limit of detection (LODabs) was determined between 5 and 10 copies of MON71800 target. The latter was not indicated by the USDA. For seed/grains the application of a sub-sampling strategy could allow detection below 0.5% but would require significant additional efforts, including the analysis of numerous sub-samples. Our tests also indicated that the duplex PCR system at the tested stage of optimisation is characterised by poor efficiency at increasing background DNA concentration in reaction. Based on the scientific evidence described in the present report, the EU-RL suggest that its testing strategy (http://gmo-crl.jrc.ec.europa.eu/GM_wheat.htm), making use of validated element and construct-specific methods and found to be more sensitive, is used to test for presence of MON71800 GM-wheat. The verified event specific method of Monsanto could be used to confirm positive findings at GM-target concentration equal or above 0.5% or it could be used for detection of GM-event MON71800 below 0.5% but it would require a costly sub-sampling strategy, which, in addition, is only possible in seeds/grains.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Cotton MON 88913 Using Real-time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the MON 88913 transformation event in cotton DNA (unique identifier MON-88913-8). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 ¿on genetically modified food and feed¿ and with Regulation (EC) No 641/2004 of 6 April 2004 ¿on detailed rules for the implementation of Regulation (EC) No 1829/2003¿, Monsanto provided the detection method and the samples: genomic DNA from cotton seeds harbouring the MON 88913 event (line ST 4664) and from conventional cotton seeds (line ST 474). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is, therefore, considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/JRC.DDG.I.4-Molecular biology and genomic

Physicochemical characterisation of gold, silica and silver nanoparticles in water and in serum-containing cell culture media

This report presents the results from a study organised under the coordination of JRC as part of a project aiming at the adaptation of the in vitro micronucleus test (Test Guideline 487) for the assessment of manufactured NMs. The aim of the first step of the project was to evaluate the physicochemical characterisation of selected representative nanomaterials (5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles) in pure water and in different complete culture media. The results of the study show that using a combination of different characterisation techniques is important to providing reliable information about the agglomeration behaviour of the tested nanoparticles in complete cell culture media (CCM). Most of the materials exhibited mild agglomeration in serum containing CCM. Only the PVP functionalised silver nanoparticles showed a size distribution change in all of the culture media that is so small that it could be attributed to solely protein adsorption without notable agglomeration. Silica nanoparticles were found to be the most sensitive to interaction with serum containing CCM, showing massive concentration and time dependent agglomeration strongly affected by the CCM composition. Extensive agglomeration might lead also to the accelerated sedimentation of the particles changing drastically the true, effective dose that the cells will receive under in vitro conditions1, 2. Thus, it has to be investigated in more detail and taken in account when designing in vitro experiments in the next phase of the project.JRC.F.2-Consumer Products Safet

In vitro cytotoxicity and cellular uptake evaluation of gold, silica and silver nanoparticles in five different cell lines: Caco-2, A549, CHO, V79 and TK6

The Joint Research Centre (JRC) is the science and knowledge service of the European Commission and provides scientific and technical support to a wide range of European Union policies. In particular, the use of manufactured nanomaterials (NMs) that are increasingly used in consumer and health products raised concerns regarding potential unintended risks to humans and the environment. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD) Working Party on Manufactured Nanoparticles (WPMN), the JRC has contributed to the development, optimisation and harmonisation of a number of test methods incl in vitro test methods suitable for risk assessment of NMs. The present report is part of an OCED project that focuses on availability of vitro methods for the assessment of the genotoxic potential of nanomaterials. An international partnership incl. the JRC aims to adapt the in vitro micronucleus test (OECD Test Guideline 487) to specific testing requirements of nanomaterials in a stepwise approach. Within the first phase of the project, the physical-chemical properties of selected nanomaterials have been intensively characterised. This second report is now focussing on the evaluation of in vitro cytotoxicity testing and the uptake of selected representative nanomaterials. 5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles have been analysed in 5 different cell lines.JRC.F.2 - Consumer Products Safet