Validation Study Report: Performance assessment of the AR-CALUX® in vitro method: to support the development of an international test guideline for Androgen Receptor Transactivation Assays (ARTA) for the detection of compounds with (anti)androgenic potential

The JRC’s EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) conducted a validation study of the AR-CALUX in vitro method. The method is applied to the detection of compounds with endocrine disrupting potential and more specifically (anti)androgen activity. The objectives of the study included assessing the reproducibility (within and between laboratories) and the relevance of the in vitro method. The participating laboratories included three test facilities from the European Union Network of Validation Laboratories for alternative methods (EU-NETVAL), being RISE, Covance, and Charles River, and, the test method submitter Biodetection Systems. The validation study report presents the results of the method performed on 46 test chemicals, evaluated for reproducibility within and between laboratories, variability within the measurements, and classification. A comparison of the classifications was made with publicly available ARTA classifications. It was concluded that this test method is reliable, has high reproducibility, low variability and merits proposal to OECD for the development of a test guideline. The statistical report and the final SOP are part of this report and can be found as separate files.JRC.F.3-Chemicals Safety and Alternative Method

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2013-April 2014)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches since the last report published in April 2013. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and international acceptance of alternative methods and approaches. R&D activities are ongoing for the complex endpoints where the toxicological processes and the mechanistic understanding have not been sufficiently elucidated yet and for which 3Rs solutions are more difficult to find. On the other hand, good progress In the validation and regulatory acceptance is made in areas where non-animal alternative methods have been developed and validated and where the focus lies in an intelligent combination/ integration of the various non-animal approaches.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2015)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches and their dissemination since the last report published in June 2014. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and regulatory/international acceptance of alternative methods and approaches and dissemination activities. R&D activities within large European or International consortia continued in toxicity areas where 3Rs solutions are more difficult to find due to the underlying complexity of the area. On the other hand, toxicity areas where promising non-animal approaches have been developed, their validation and regulatory acceptance/international adoption could be progressed. Particular emphasis was given to the best and most intelligent combination and integration of these different non-animal approaches to ultimately obtain the required information without resorting to animal testing.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2016)

Replacement, Reduction and Refinement of animal testing is anchored in EU legislation. Alternative non-animal approaches facilitate a shift away from animal testing. Cell-based methods and computational technologies are integrated to translate molecular mechanistic understanding of toxicity into safety testing strategies.JRC.F.3-Chemicals Safety and Alternative Method

Transposon mmutagenese van het genoom van Azospirillum brasilense

SIGLEKULeuven Campusbibliotheek Exacte Wetenschappen / UCL - Université Catholique de LouvainBEBelgiu

LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information, especially when evidence suggests the presence of unauthorized GMO on the market, requires efficient and in the later situation also rapid approaches. Here we report on the optimization of a restriction independent method named 'Rapid Amplification of genomic DNA Ends' (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, we applied the same methodology to report on the DNA sequences surrounding the insert of GM rice event LLRICE62.JRC.I.3-Molecular Biology and Genomic

Supporting the Implementation of the EU Community Strategy on Endocrine Disrupters

The regulatory challenge posed by endocrine disrupting chemicals cuts across several major pieces of EU legislation, either already in force such as REACH, the Plant Protection Products Regulation and the new Cosmetics Regulation, or still at the stage of a proposal, such as the revision of the Biocides Directive. The EU Community Strategy on Endocrine Disrupters provides a valuable coordination framework to develop a systematic approach for the identification and assessment of endocrine disruptors, which can be applied across the different pieces of legislation. The European Commission’s Joint Research Centre is involved in a number of scientific/technical activities such as the validation of new testing methods, the development of a web based IT platform for endocrine active substances and the development of an OECD harmonised template for reporting on intermediate toxicological effects. The aim of these policy support activities is to provide tools and scientific knowhow that will reduce the number of animal experiments needed to assess potential endocrine disrupting properties of chemicals while at the same time ensuring the maximum protection for human health.JRC.I.5-Systems Toxicolog

Assessing Copy Number of MON810 Integrations in Commercial Seed Maize Varieties by 5' Event-specific Real-time PCR Validated Method Coupled to 2-deltadelta CT Analysis

The objective of the present study was to assess the applicability of the MON 8105' event-specific method validated by the Community Reference Laboratory for Genetically Modified Food and Feed(CRL-GMFF) that is commonly used for quantitative purposes. This 5' event-specific /hmg-taxon gene real-time PCR protocol coupled to 2 analysis was the chosen approach to determine the MON 810 insert copy number per haploidgenome across 26 genetically modified (GM) commercial maize varieties. Variety DK513 containing one copy integration per haploid genome was used as calibrator in each assay. Complementary data from end-point real-time PCRs that targeted specifically the MON 810 insert were also analysed. Global results assessed and guaranteed the genetic intactness of the transgenic integration per haploid genome for 24 out of the 26 commercial varieties studied,which showed no significant differences between 2 values respect to the calibrator value. Conversely,two varieties showed no transgenic insert in their genomes. This validated analytical method was suitable for MON 810 detection and quantification purposes.JRC.I.3-Molecular Biology and Genomic