PubMed Health⌕ Search

Biomedical subjects

Horst Spielmann

Publications and source records attributed to Horst Spielmann.

At least 37 records · Page 2Linked to original sources
PubMed2004

Validation of the postimplantation rat whole-embryo culture test in the international ECVAM validation study on three in vitro embryotoxicity tests.

A detailed report is presented on the performance of the postimplantation rat whole-embryo culture (WEC) test in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the WEC test can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.

Animal Testing Alternatives↗
PubMed2004

Trends in improving the embryonic stem cell test (EST): an overview.

The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the teratogenic and embryotoxic potential of drugs and chemicals. It is based on the capacity of murine ES cells (cell line D3) to differentiate into contracting myocardial cells under specific cell culture conditions. The appearance of beating cardiomyocytes in embryoid body (EB) outgrowths is used as a toxicological endpoint to assess the embryotoxic potential of a test substance. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study the EST predicted the embryotoxic potential of chemicals and drugs with the same reliability as two other in vitro embryotoxicity tests, which employed embryonic cells and tissues from pregnant animals. In a joint research project with German pharmaceutical companies we have successfully improved the EST by establishing molecular endpoints of differentiation in cultured ES cells. The quantification of cardiac-specific protein expression by intracellular flow cytometry has been studied in the presence of chemicals of different embryotoxic potential. The results obtained using molecular endpoints specific for differentiated cardiomyocytes employing FACS (fluorescence-activated cell sorting) analysis will be presented in comparison to the validated endpoint - the microscopic analysis of beating areas. FACS analysis provides a more objective endpoint for predicting the embryotoxic potential of chemicals than the validated method. Furthermore, flow cytometry promises to be suitable for high-throughput screening systems (HTS). In addition, our partners from the joint project have improved the EST by developing protocols that stimulate differentiation of ES cells into neural and endothelial cells, chondrocytes and osteoblasts, because some substances might have embryotoxic effects on specific cell-types other than cardiomyocytes. These protocols have been successfully established at ZEBET and in the participating laboratories. Additionally, molecular endpoints have been established for the detection of specific differentiation pathways. Furthermore, new prediction models (PMs) have been developed using single endpoints of the EST.

Animal Testing Alternatives↗
PubMed2004

Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests.

An ECVAM-funded prevalidation study (PV) was conducted during 1999 and 2000 to identify in vitro tests capable of reliably distinguishing between skin irritants (I) and non-irritants (NI) according to European Union risk phrases ("R38" or no classification). The tests evaluated were EpiDerm, EPISKIN, PREDISKIN, the non-perfused pig ear method, and the mouse skin integrity function test (SIFT). Whereas reproducibility of the two human skin model tests and SIFT was acceptable, none of the methods was deemed ready to enter a formal validation study due to their low predictivity. The ECVAM Skin Irritation Task Force therefore suggested improvements of protocols and prediction models for these tests. Furthermore, it was agreed that experience gained with the two human-skin models be shared, and a common protocol should be developed for EpiDerm and EPISKIN (Zuang et al., 2002). When we applied an improved EPISKIN protocol (Portes et al., 2002) to the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was far too low. In 2003, the EPISKIN protocol was further refined by extension of the post-incubation period following chemical exposure. In the current study, we evaluated this EPISKIN refinement by applying it to EpiDerm. In addition, we developed technical improvements for the application of the test chemicals and rinsing procedure, which reduced the variability of results and increased the percentage of correct predictions. A set of twenty non-coded reference substances from the ECVAM prevalidation study phase III (Fentem et al., 2001) was tested with the final protocol in three independent runs. Both high sensitivity (80%) and high specificity (78%) were achieved, and the statistical probability of correct classifications was high, so that the test is now regarded ready for formal validation.

Animal Testing Alternatives↗
PubMed2004

Report and recommendations of the international workshop "Retrieval approaches for information on alternative methods to animal experiments".

In the member states of the EU and in the USA, scientists are obliged by animal welfare legislation not to conduct an animal experiment if another scientifically satisfactory method is reasonably and practicably available. To meet the regulatory obligation to use alternatives to animal experiments, scientists should consult literature and other relevant sources on alternatives prior to any experimental study on laboratory animals. It is the responsibility of the individual scientist to select the most appropriate database to obtain information on alternatives, which have been defined as methods that refine, reduce or replace animal experiments (the 3 Rs concept of Russell and Burch (1959)). Specialised information services provide support to scientists searching for publications on alternative methods. On occasion of a workshop in Berlin in November 2003, representatives of animal welfare information centres discussed currently available information sources on alternative methods, index terms for alternative methods, and search strategies based on index terms for alternative methods. ZEBET presented an investigation on the current status of indexing systems on alternative methods in established literature databases. The project analysed how the results of a search for publications on alternatives was influenced by the indexing procedure. The results of the study were exemplified by a typical search result. The results of the study indicated that the current indexing systems do not provide the required information, since not all of the relevant information is indexed under "alternative methods". The workshop participants developed recommendations for ad hoc working groups and research projects, e.g. development of suitable search strategies on alternative methods for scientists.

Animal Testing Alternatives↗
PubMed2004

Third EU report on the statistics on animals used for experimental and other scientific purposes: trends, problems, conclusions.

A brief overview is given of the data published in the third report on the number of animals used for scientific purposes in the European Union (EU), in 1999. In comparison to the second EU statistical report, the meaningfulness of the data has been greatly improved, since 14 EU Member States have now used the "EU tables". Even though the total number of animals officially reported in the EU for 1999 (9.81 million animals) is lower than the number reported for 1996 in the second report (11.65 million animals), an increase in animal numbers was observed in several Member States. Repeatedly, the use of transgenic animals was considered to be the cause for this. Most national authorities provided some comments on the data they submitted. However, the quality of these accompanying notes varies considerably, with the comments provided by The Netherlands, Finland, Sweden, Austria, Denmark and the United Kingdom containing the most-detailed remarks on their data.

Animal Experimentation↗
PubMed2003

[Third statistical report on the numbers of laboratory animals used in the European Union--tendencies, problems, decisions].

In 2003, the European Commission has published its third statistical report on the numbers of laboratory animals used in the European Union, in which the data of 1999 are analysed. For the first time, with the exception of Germany, the data presented were raised on the basis of a new registration scheme. The significance of these data is limited due to deficiencies of the tables used, restricted comparability with previously raised data, and lacking relevance to the current situation. Despite this imperfect basis, the European Commission concludes in its report that there has been a significant reduction in the number of laboratory animals used in the EU compared to the previous statistics. This statement must be regarded critically as several national statistics of the last years verify that the number of laboratory animals used has in fact increased. According to the comments of various national authorities included in the report, particularly genetic engineering is responsible for the increase in animal numbers.

Animal Welfare↗
PubMed2003

The future of animal experiments in nutrition research.

An overview is given on the legal framework in Europe for the use of experimental animals set by EU (European Union) Directive 86/609/EEC and on the activities of EU member states to implement this directive for regulatory testing in animals. Recent progress in implementing the 3Rs concept of Russel and Burch into regulatory guidelines of the EU are described. Moreover the validation of non-animal tests to be used for regulatory purposes is outlined. From the regulatory and the animal welfare perspective the international harmonisation of test guidelines and the mutual acceptance of data are the way forward for chemical safety testing including food additives and contaminants. Examples are given for the replacement of diagnostic animal tests by analytical test methods, e.g. hormones, Clostridium botulinum toxins and neurotoxins from algae in shellfish.

Animal Experimentation↗
PubMed2002

Currently available in vitro methods used in the regulatory toxicology.

About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in regulatory toxicology. For reasons of animal welfare and better science, many validation studies of various in vitro methods were performed between 1985 and 1995 for their potential to replace the Draize eye irritation test. Albeit the fact that four in vitro methods (HET-CAM, BCOP, IRE, and ECE) have gained regulatory tolerance in Europe for the classification of severe eye irritants, the lessons learned mainly from these validation studies led to the definition of internationally harmonised OECD validation principles and acceptance criteria (Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods, 1996. OECD Publication Office, Paris, France). Application of these principles to prevalidation and validation studies, and additional special studies performed in Europe with the support of ECVAM, ended in the scientific validation of new in vitro methods for the prediction of skin corrosivity and phototoxicity. They were accepted for regulatory use in Europe and adopted on 8 June 2000 as test methods B. 40 and B. 41 of Annex V of Directive 67/548/EEC. In quite a different approach, European industry has submitted in-house validation data to support a Draft OECD Test Guideline for in vitro dermal absorption testing, which-after peer review and a long lasting international discussion-will now be adopted by the OECD. The increasing importance of regulatory measures derived from quantitative cytotoxicity tests, and some examples of regulatory accepted in vitro methods, where the specific purpose is restricted to a specific regulation (e.g. testing of medicinal products, or medical devices), are also addressed.

Animal Testing Alternatives↗
PubMed2002

Animal use in the safety evaluation of chemicals: harmonization and emerging needs.

An overview is provided of the legal framework in Europe for the use of experimental animals set by European Union (EU) Directive 86/609/EEC and of the activities of EU member states to implement this directive for regulatory testing in animals. The use of animal data in the safety assessment of chemicals by services of the EU Commission and by EU member states is described. Specific examples are given for the current use of animal tests in Europe for the safety evaluation of industrial chemicals, pesticides and biocides, cosmetics, endocrine disrupters, and existing chemicals. Recent progress in implementing the 3Rs concept of Russell and Burch into regulatory guidelines of the EU are described, with particular reference to acute systemic and local toxicity testing. Progress in implementing the 3Rs concept in the EU is compared with the situation outside Europe, particularly with the incentive initiated by the Organisation for Economic Co-operation and Development, to reduce regulatory testing in animals. The harmonization of toxicity test guidelines initiated by the Organisation for Economic Co-operation and Development in 1982 has been the most successful measure to reduce pain and distress of laboratory animals in regulatory testing. From the animal welfare perspective, the international harmonization of test guidelines and the mutual acceptance of data are, therefore, the way forward for all areas of chemicals testing.

Animal Welfare↗
PubMed2002

Validation successes: chemicals.

The ECVAM validation concept, which was defined at two validation workshops held in Amden (Switzerland) in 1990 and 1994, and which takes into account the essential elements of prevalidation and biostatistically defined prediction models, has been officially accepted by European Union (EU) Member States and by the Federal regulatory agencies of the USA and the OECD. The ECVAM validation concept was introduced into the ongoing ECVAM/COLIPA validation study of in vitro phototoxicity tests, which ended successfully in 1998. The 3T3 neutral red uptake in vitro phototoxicity test was the first experimentally validated in vitro toxicity test recommended for regulatory purposes by the ECVAM Scientific Advisory Committee (ESAC). It was accepted by the EU into the legislation for chemicals in the year 2000. From 1996 to 1998, two in vitro skin corrosivity tests were successfully validated by ECVAM, and they were also officially accepted into the EU regulations for chemicals in the year 2000. Meanwhile, in 2002, the OECD Test Guidelines Programme is considering the worldwide acceptance of the validated in vitro phototoxicity and corrosivity tests. Finally, from 1997 to 2000, an ECVAM validation study on three in vitro embryotoxicity tests was successfully completed. Therefore, the three in vitro embryotoxicity tests, the whole embryo culture (WEC) test on rat embryos, the micromass (MM) test on limb bud cells of mouse embryos, and the embryonic stem cell test (EST) including a permanent embryonic mouse stem cell line, are considered for routine use in laboratories of the European pharmaceutical and chemicals industries.

3T3 Cells↗
PubMed2002

[White Paper of the European Commission--strategy for a new chemicals policy].

The current chemicals regulation in EU member states provides sufficient data on new chemicals but almost non on the about 100.000 existing chemicals. To improve the situation the EU Commission is proposing in a recent White Paper on a strategy for a new chemicals policy to regulate new and existing chemicals in an identical manner. The Commission suggests to use in vitro methods to provide information rapidly on hazardous health and environmental properties of chemicals. The fundamental change in the testing strategy, which is relying on in vitro methods rather than testing in animals is most welcome not only from the scientific and economic point of view but also from the perspective of the protection of animals and the environment. A calculation of the costs and time for testing all existing chemicals in animal experiments shows that this task is unrealistic from a financial point of view and also when taking into account the time required for testing. Taking into account the proposals provided by several working groups of the EU validation centre ECVAM during the past year, a concept is described that will allow the testing of all existing chemicals applying only non-animal tests. Since the new testing strategy will be cheaper and faster it will be more acceptable from both the financial and the ethical point of view. Finally, establishing the proposed new in vitro tests in the safety testing strategy will require considerable funding of validation studies by the EU Commission.

Animal Testing Alternatives↗
PubMed2002

[Improving the embryonic stem cell test (EST) by establishing molecular endpoints of tissue specific development using murine embryonic stem cells (D3 cells)].

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. In the embryonic stem cell test (EST) the capacity of ES cells of the mouse cell line D3 to differentiate into contracting cardiomyocytes is used to assess the embryotoxic potential of test compounds and in addition, the effects on the viability of ES cells and differentiated mouse fibroblasts (cell line 3T3) are compared. The three endpoints are used to classify the embryotoxic potential of chemicals after 10 days of exposure: (i) the inhibition of differentiation of ES cells into cardiomyocytes (ID50) and (ii) the decrease of viability of 3T3 cells (IC503T3) and (iii) ES cells (IC50D3) in a MTT cytotoxicity test. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study funded by ECVAM it could be demonstrated that the EST can predict the embryotoxic potential of a test compound as good as frequently used mammalian systems based on pregnant animals. In a joint project with major German pharmaceutical companies we are attempting to improve the EST by establishing molecular endpoints of differentiation (e.g. cardiac, neuronal, chondrogenic) in cultured ES cells. We have studied the expression of tissue specific proteins in ES cell cultures in the presence of embryotoxic chemicals by immunofluorescent antibody techniques, e.g. FACS analysis. The other groups are focusing on endogenous gene expression in early development by RT-PCR methods or the DNA microarray technique. The results obtained recently using molecular markers specific for cardiac differentiation and employing intracellular flow cytometry for quantification will be presented. Molecular endpoints will allow improvement of the EST by measuring gene expression patterns in a small number of murine ES cells.

3T3 Cells↗
PubMed2002

A chair on alternatives?

An overview is given on the legal framework in Europe for the use of experimental animals set by EU (European Union) Directive 86/609/EEC and on the activities of EU member states to implement this directive in the field of regulatory testing in animals. The significant decrease in the number of experimental animals in Germany during the past decade is described with particular reference to the recent increase that is due to transgenic animal models. From the regulatory and the animal welfare perspective the international harmonisation of test guidelines and the mutual acceptance of data are the way forward for chemical safety testing. The recent White Paper of the EU Commission for the future chemicals policy calls for an immediate increase in the number of validated in vitro toxicity tests to be accepted for regulatory purposes in the EU. In addition, deficits of properly educating young scientists in Germany in conducting animal experiments and implementing the 3-Rs concept of Russel and Burch are described. It is therefore, quite urgent to establish new chairs on animals and alternatives at universities in Europe. They should focus on both education young students of the biomedical sciences in the humane use of laboratory animals according to the 3-Rs concept and on developing new toxicity tests to be validated for regulatory purposes under the new EU chemicals policy by the established validation centres in Europe.

Animal Rights↗
PubMed1998

[Reduction and replacement of animal tests required in standards for safety testing]

Animal tests which have to be performed according to national (DIN) and international standards (EN and ISO) are evaluated according to the 3Rs principle of Russel and Burch in order to reduce suffering and numbers of test animals and also to completely replace them by non-animal methods. When on January 1, 1995, the EU Medical Devices Directive 93/42/EEC was accepted by all EU member states, animal tests that were essential parts of DIN, EN and ISO standards got a new legal status since for biocompatibility testing EU Directive 93/42/EEC is refering to these standards. In the present study all DIN standards were collected, in which animal testing is required, and they were then evaluated from the view points of both scientific state of the art and of consumer protection to decide if these animal tests can be replaced by non-animal methods. Our search of the literature revealed not only that to each of the animal tests promising alternatives have been proposed but also that only a few of them have sufficiently been validated to date. Moreover, we found that several of the animal tests described in DIN standards are not any more representing the current state of the art in the biomedical sciences. Therefore, these animal tests should immediately be deleted from the standards. Thus we are proposing to the DIN institue, the national agency for standards in Germany, immediate action which is required to update each of these DIN standards according to EU Directive 86/609/EEC for the protection of experimental animals. Our proposals are recommending to completely delete some of the animal tests from the standards, or to replace them by validated alternatives or to conduct formal validation studies with the most promising alternatives.

Journal Article↗
PubMed1998

[Results of the first phase of the ECVAM project "prevalidation and validation of three in vitro embryotoxicity tests"]

The results of the experimental prevalidation of three in vitro embryotoxicity tests in an ECVAM study are reported: the rat whole embryo culture assay (WEC), the micromass assay (MM) and the embryonic stem cell test (EST). Three test chemicals assigned to the three classes of embryotoxicity, non-, moderate- and strong embryotoxic, and also a positive and a negative control chemical were repeatedly tested in each test in two laboratories. The data obtained were evaluated independently by a biostatistician. It could be shown that the standard protocols (SOPs) for the three in vitro embryotoxicity tests could be transferred to a second laboratory and that reproducible results were obtained. The three methods were able to discriminate the test chemicals according to their embryotoxic potential. In addition, within the scope of this study, a test chemical database for in vitro embryotoxicity testing was established that will be used in the following formal validation of the three in vitro tests.

Journal Article↗
PubMed1998

[ERGATT/ECVAM Workshop on Acceptance of Validated Alternative Methods: Amden III]

Since validated in vitro toxicity tests have so far not been accepted by regulators the third international ERGATT/ECVAM validation workshop was held from January 26-30, 1998, in Amden/Switzerland. Scientific and political reasons for a delay of the acceptance of alternative methods were identified in the areas of phototoxicity, skin penetration and skin corrosivity and strategies to promote the acceptance were suggested. The OECD will harmonise the formal of criteria for the regulatory acceptance of new experimentally validated tests and of established tests from industry for which in-house and literature data exist. In vitro tests for phototoxicity and skin penetration will be accepted shortly by the OECD. For skin corrosivity two in vitro tests will be put forward to the OECD later this year. The Amden III workshop suggests that, the same acceptance criteria must be used for animal and in vitro tests. Finally, the OECD should remove tests that cause pain and distress to test animals from the official OECD list of official test guidelines if validated alternative methods exist. When obsolete animal tests are taken from the list of official OECD test guidelines, they will not any longer be accepted at the international level. Thus, the third Amden workshop has contributed new concepts for the regulatory acceptance of alternative toxicity tests which are supported by regulators in Europe, the USA and the OECD.

Journal Article↗