Towards the development of an in vitro assay to predict osteotoxicity in the developing embryo: an approach

To assess the adverse effect of a substance on the developing embryo, animal experimentation is employed. Approximately half of the tested compounds are found to cause detrimental skeletal damage. Therefore, an in vitro assay with the capacity to predict developmental osteotoxicity would be a highly valuable tool to reduce animal expenditure.Their capacity for self-renewal combined with their unique differentiation potential has led to embryonic stem cells (ESCs) becoming an attractive model system to study developmental processes.The fact that differentiating ESCs recapitulate early stages of mammalian development has made them a pivotal screening tool to identify potentially embryotoxic substances.The ZEBET laboratory uses both murine and primate embryonic stem cells as models to study osteogenic differentiation on a molecular level. Expression profiling of marker genes/proteinsis employed to identify and establish molecular endpoints indicative of skeletal damage during embryogenesis.A detailed description of the research strategy and preliminary results will be presented at the meeting

Human Multipotent Progenitors — A Promising Cell Model for Assessing Developmental Osteotoxicity In Vitro

To assess the potential adverse effects of substances on bone development in man, animalexperimentation is commonly employed. In vivo testing is labour- and cost-intensive andrequires a high number of laboratory animals. Up to now, there exists no validated alternativemethod to assess developmental bone toxicity in vitro. The derivation of multipotent progenitorswith mesenchymal characteristics from human embryonic stem cells (hES-MPs)constitutes one strategy in regenerative medicine, to work with a cell source that exhibits alow risk of tumour formation after transplantation. These cells display the typical morphologyof primary human mesenchymal stem cells and show a similar gene expression profile. Inaddition, they have a high proliferative activity and the capacity to differentiate into specialisedcell types of mesenchymal origin in vitro (adipocytes, chondrocytes, osteoblasts).Notably, their experimental use in Germany is exempt from regulatory approval enforced bythe German Stem Cell Act. Therefore, hES-MPs appear to be an attractive and promisinghuman-based cell model to screen for potential osteotoxic substances. Crucial stages duringosteogenesis involve the proliferation of progenitor cells, followed by their gradual differentiationinto functional osteoblasts, the maturation of the extracellular matrix (ECM) and, ultimately,the mineralisation of the ECM. Initial work to study the osteogenic differentiationprocess of hES-MPs has already been accomplished by another research group. Based on theirfindings, we further characterised the differentiation process regarding the influence of differentinducer cocktails, the delineation of the developmental stages and the expression of lineage-specific protein markers. A diverse range of biochemical and molecular biologicalmethods, e.g. cell viability and proliferation assays, colorimetric assays, cytochemical stainings,flow cytometry and western blotting, were employed to monitor the underlying molecularprocesses. Additionally, we performed initial experiments to investigate the sensitivity ofthe differentiating cells toward developmental toxicants. In summary, the hES-MP cell modelmight prove to be a valuable tool for assessing compound-mediated adversity on human bonedevelopment in vitro

Toxicological endpoints to assess developmental bone toxicity in vitro

To predict the toxic potential of industrial chemicals and pharmaceutical products on human bone development, rodent and non-rodent (e.g. rabbits) in vivo models are commonly used.The assessment of osteogenic toxicity in the embryo is integrated into the OECD testing guideline #414, which covers prenatal developmental toxicity. In experiments using rodents, onehalf of the litter (approximately 450 animals per test substance) is sacrificed for the examination of skeletal damage. In addition to the high number of laboratory animals required, these invivo studies are costly and time-consuming. Until today, there is no validated in vitro test to assess developmental bone toxicity.Therefore, current work at ZEBET in the frame of a joint project, funded by the German Ministry for Education and Research (BMBF), focuses on the development of a robust assay with the capacity to predict osteotoxicity in the embryo.Embryonic stem cells are pluripotent cells which can differentiate into a multitude of diverse cell types. Their capacity for unlimited self-renewal together with their ability to faithfullyrecapitulate early developmental programmes in the embryo in vitro makes them an extremely attractive model for embryonic toxicity studies.Crucial stages during osteogenesis involve the sequential expression of a tightly regulated set of molecular markers and the mineralisation of the extracellular matrix. We are currentlyassessing whether any of these markers can serve as a predictive endpoint to assess bone toxicity using a diverse range of molecular biological methods, e.g. real-time PCR, Western blot,flow cytometry, Fourier transform infrared spectroscopy (FTIR) and cytochemical staining. Mouse embryonic stem cells (line D3) are currently employed as a model system to study osteogenesis.As a next step this approach will be expanded to other cellular systems including stem cells from rhesus monkey, human iPS cells as well as human mesenchymal stem cells. The applicability of the different molecular markers and themorphological approach depending on the method is discussed regarding their potential as toxicological endpoint

Using the Go3R Search Engine to Collect Toxicological Data and Three Rs-relevant Information for REACH Registration Dossiers

The European REACH Regulation No. 1907/2006 on the Registration, Evaluation and Authorisationof Chemicals obliges companies to register all chemicals manufactured or imported inquantities above 1 tonne per year. Since detailed information on the intrinsic properties of chemicalsubstances has to be provided in the registration dossiers, comprehensive collection of allexisting relevant data serves to avoid animal testing. Also, when new data have to be compiledto obtain hazard information, testing on vertebrates may only be undertaken as a last resort.Hence, meeting the challenges of the REACH legislation further requires the continuous updatingof the information on newly-developed test methods that replace, reduce and refine animaltesting. The knowledge-based search engine Go3R (www.Go3R.org) has been designed to assistscientists from industry and regulatory authorities in accomplishing both tasks. Analysing the22 million documents of the PubMed and Toxicology Data Network (TOXNET) databases, Go3Rmakes use of expert knowledge on Three Rs methods and regulatory toxicology, laid down in anetwork of concepts, terms and synonyms, to recognise the contents of documents. Searchresults are automatically sorted into a table of contents, which is displayed alongside the list ofdocuments retrieved. By clicking onto different sections of the table of contents, the user canquickly filter the set of documents by topics of interest. Documents containing hazard informationare automatically assigned to the IUCLID5 toxicological information section of the table ofcontents that follows the endpoint-specific IUCLID5 categorisation required for REACH registrationdossiers. To enable Go3R to sort documents by the toxicological endpoint being investigated,complex endpoint-specific search queries were compiled and integrated into the searchengine. Dependent upon the endpoint, up to 100% of relevant documents containing either invitro or in vivo hazard information were correctly sorted into the respective IUCLID5 category.Since Go3R can be customised to search in different databases, the IUCLID5 categorisation isalso applicable to the screening and sorting of unpublished in-house data, and it can also be usedto group substances. The Three Rs specific searches section of the table of contents recognisesThree Rs-relevant documents and sorts these by Three Rs-specific information. The table of contentsprovides an overview on available test methods that replace, reduce or refine animal testing\ sorted by area of use, types of cells or tissues used in in vitro systems, in vitro endpointsand in vitro endpoint detection methods. Since important information on Three Rs alternativesis published not only in scientific journals, but also on dedicated internet sites, Go3R furtherallows searching the entire world-wide-web employing a Google-based search tool with priorrankingof information from Three Rs relevant websites. This option supports the retrieval ofinformation on the status of validation and regulatory acceptance of specific test methods or theirStandard Operating Procedure. The presentation will provide examples on how to use Go3R toretrieve substance-specific toxicological data and Three Rs relevant documents and will discussthe ongoing evaluation of the Go3R search engine

Using the Go3R Search Engine to Collect Toxicological Data and Three Rs-relevant Information for REACH Registration Dossiers

The European REACH Regulation No. 1907/2006 on the Registration, Evaluation and Authorisationof Chemicals obliges companies to register all chemicals manufactured or imported inquantities above 1 tonne per year. Since detailed information on the intrinsic properties of chemicalsubstances has to be provided in the registration dossiers, comprehensive collection of allexisting relevant data serves to avoid animal testing. Also, when new data have to be compiledto obtain hazard information, testing on vertebrates may only be undertaken as a last resort.Hence, meeting the challenges of the REACH legislation further requires the continuous updatingof the information on newly-developed test methods that replace, reduce and refine animaltesting. The knowledge-based search engine Go3R (www.Go3R.org) has been designed to assistscientists from industry and regulatory authorities in accomplishing both tasks. Analysing the22 million documents of the PubMed and Toxicology Data Network (TOXNET) databases, Go3Rmakes use of expert knowledge on Three Rs methods and regulatory toxicology, laid down in anetwork of concepts, terms and synonyms, to recognise the contents of documents. Searchresults are automatically sorted into a table of contents, which is displayed alongside the list ofdocuments retrieved. By clicking onto different sections of the table of contents, the user canquickly filter the set of documents by topics of interest. Documents containing hazard informationare automatically assigned to the IUCLID5 toxicological information section of the table ofcontents that follows the endpoint-specific IUCLID5 categorisation required for REACH registrationdossiers. To enable Go3R to sort documents by the toxicological endpoint being investigated,complex endpoint-specific search queries were compiled and integrated into the searchengine. Dependent upon the endpoint, up to 100% of relevant documents containing either invitro or in vivo hazard information were correctly sorted into the respective IUCLID5 category.Since Go3R can be customised to search in different databases, the IUCLID5 categorisation isalso applicable to the screening and sorting of unpublished in-house data, and it can also be usedto group substances. The Three Rs specific searches section of the table of contents recognisesThree Rs-relevant documents and sorts these by Three Rs-specific information. The table of contentsprovides an overview on available test methods that replace, reduce or refine animal testing\ sorted by area of use, types of cells or tissues used in in vitro systems, in vitro endpointsand in vitro endpoint detection methods. Since important information on Three Rs alternativesis published not only in scientific journals, but also on dedicated internet sites, Go3R furtherallows searching the entire world-wide-web employing a Google-based search tool with priorrankingof information from Three Rs relevant websites. This option supports the retrieval ofinformation on the status of validation and regulatory acceptance of specific test methods or theirStandard Operating Procedure. The presentation will provide examples on how to use Go3R toretrieve substance-specific toxicological data and Three Rs relevant documents and will discussthe ongoing evaluation of the Go3R search engine

Using the Go3R Search Engine to Collect Toxicological Data and Three Rs-relevant Information for REACH Registration Dossiers

The European REACH Regulation No. 1907/2006 on the Registration, Evaluation and Authorisationof Chemicals obliges companies to register all chemicals manufactured or imported inquantities above 1 tonne per year. Since detailed information on the intrinsic properties of chemicalsubstances has to be provided in the registration dossiers, comprehensive collection of allexisting relevant data serves to avoid animal testing. Also, when new data have to be compiledto obtain hazard information, testing on vertebrates may only be undertaken as a last resort.Hence, meeting the challenges of the REACH legislation further requires the continuous updatingof the information on newly-developed test methods that replace, reduce and refine animaltesting. The knowledge-based search engine Go3R (www.Go3R.org) has been designed to assistscientists from industry and regulatory authorities in accomplishing both tasks. Analysing the22 million documents of the PubMed and Toxicology Data Network (TOXNET) databases, Go3Rmakes use of expert knowledge on Three Rs methods and regulatory toxicology, laid down in anetwork of concepts, terms and synonyms, to recognise the contents of documents. Searchresults are automatically sorted into a table of contents, which is displayed alongside the list ofdocuments retrieved. By clicking onto different sections of the table of contents, the user canquickly filter the set of documents by topics of interest. Documents containing hazard informationare automatically assigned to the IUCLID5 toxicological information section of the table ofcontents that follows the endpoint-specific IUCLID5 categorisation required for REACH registrationdossiers. To enable Go3R to sort documents by the toxicological endpoint being investigated,complex endpoint-specific search queries were compiled and integrated into the searchengine. Dependent upon the endpoint, up to 100% of relevant documents containing either invitro or in vivo hazard information were correctly sorted into the respective IUCLID5 category.Since Go3R can be customised to search in different databases, the IUCLID5 categorisation isalso applicable to the screening and sorting of unpublished in-house data, and it can also be usedto group substances. The Three Rs specific searches section of the table of contents recognisesThree Rs-relevant documents and sorts these by Three Rs-specific information. The table of contentsprovides an overview on available test methods that replace, reduce or refine animal testing\ sorted by area of use, types of cells or tissues used in in vitro systems, in vitro endpointsand in vitro endpoint detection methods. Since important information on Three Rs alternativesis published not only in scientific journals, but also on dedicated internet sites, Go3R furtherallows searching the entire world-wide-web employing a Google-based search tool with priorrankingof information from Three Rs relevant websites. This option supports the retrieval ofinformation on the status of validation and regulatory acceptance of specific test methods or theirStandard Operating Procedure. The presentation will provide examples on how to use Go3R toretrieve substance-specific toxicological data and Three Rhe ongoing evaluation of the Go3R search engine