Stability recovery performance in adults over a wide age range: A multicentre reliability analysis using different lean-and-release test protocols.

The ability to effectively increase the base of support is crucial to prevent from falling due to stability disturbances and has been commonly assessed using the forward-directed lean-and-release test. With this multicentre study we examined whether the assessment of stability recovery performance using two different forward lean-and-release test protocols is reliable in adults over a wide age range. Ninety-seven healthy adults (age from 21 to 80 years) were randomly assigned to one out of two lean angle protocols: gradual increase to maximal forward-lean angle (maximal lean angle; n = 43; seven participants were excluded due to marker artefacts) or predefined lean angle (single lean angle; n = 26; 21 participants needed to be excluded due to multiple stepping after release or marker artefacts). Both protocols were repeated after 0.5 h and 48 h to investigate intra- and inter-session reliability. Stability recovery performance was examined using the margin of stability at release (MoSRL) and touchdown (MoSTD) and increase in base of support (BoSTD). Intraclass correlation coefficients (confidence intervals at 95%) for the maximal lean angle and for the single lean angle were respectively 0.93 (0.89-0.96) and 0.94 (0.89-0.97) in MoSRL, 0.85 (0.77-0.91) and 0.67 (0.48-0.82) in MoSTD and 0.88 (0.81-0.93) and 0.80 (0.66-0.90) in BoSTD, with equivalence being revealed for each parameter between all three measurements (p < 0.01). We concluded that the assessment of stability recovery performance parameters in adults over a wide age range with the means of the forward lean-and-release test is reliable, independent of the used lean angle protocol.

Electrophysiological predictors of hearing deterioration based on AEP monitoring during petroclival meningioma resection.

The objective of this study was to calculate the risk of postsurgical hearing deterioration as a function of changes in the amplitude and latency of the most stable components (waves III and V) of the auditory evoked potential (AEP) during petroclival meningioma resection surgery. We retrospectively analyzed intraoperative AEP monitoring results and pre- and postsurgical hearing status in 40 consecutive patients who were surgically treated for petroclival meningiomas. Statistical analyses were conducted to identify the most sensitive and specific way to predict hearing dysfunction after surgery. Patients' mean age was 59 ± 10 years, and 31 (77.5%) were women. Twelve (30%) patients presented with clinically detectable hearing impairment preoperatively. At the first postoperative assessment, four of those 12 patients reported subjective improvement, and eight reported hearing deterioration. Of those eight, four remained stable and four recovered hearing by the last assessment. Wave III latency reached its highest specificity (100%) and sensitivity (71.43%) at x = 143%. Wave V latency, on the other hand, reached its highest sensitivity (71%) and specificity (93%) at x = 124%. Finally, wave V amplitude reached its highest sensitivity (100%) and specificity (79%) at x = 74%. Intraoperative alterations of wave III latency and wave V amplitude seem to be highly sensitive and specific at predicting the risk of auditory dysfunction in patients undergoing petroclival meningioma resection and should be used to determine maximum resection with preservation of function.

Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I).

We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85-95% and a between-laboratory-reproducibility of 90%.

Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (µM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.

Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

Development of an in vitro modified skin absorption test for the investigation of the follicular penetration pathway of caffeine.

The Organization for Economic Cooperation and Development (OECD) recommends caffeine as a reference substance for in vitro skin absorption tests using Franz diffusion cells (FDC). However, it has not been possible to investigate the follicular penetration pathway using this method until now. The aim of this study was to develop a technique to allow the examination of the follicular penetration pathway of a substance penetrating into the skin. The OECD standard method was therefore combined with the follicle closing technique (FCT), an established in vivo method. By using test skin of varying follicular densities, different penetration values were obtained for the test substance caffeine. The follicular penetration rate was determined by an indirect calculation after modifying the in vivo FCT for use in the in vitro FDC. This method is the first to allow the differentiation of penetration pathways by combining the OECD standard method (using the FDC) and the FCT. Caffeine showed a surprisingly high rate of penetration through the follicular shunts in vitro.

Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests.

The aim of this study was to clarify the differences in the phototoxicity of bergamot oil obtained from four different suppliers. Spectral and chemical analyses were performed to identify presence of photoactive compounds in the test samples. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D PT). Confirmatory photopatch tests in a group of volunteers were performed using the first non-phototoxic concentration determined in the H3D PT. The spectral and chemical analyses revealed, that two samples of bergamot oil exhibited a potential for photoactivation. These oils were subsequently classified as phototoxic in the 3T3 NRU PT, however, only on the basis of borderline results and depending on the solvent used. H3D PT revealed clear classifications, correlating well with the findings of spectral and chemical analysis. The test was, however, not yet capable of precise prediction of safe, non-phototoxic concentrations. Additional endpoints, e.g. interleukin determination might be employed to increase the sensitivity of the test. Although the study showed the usefulness of the tiered testing strategy, currently, the extrapolation of in vitro results to human situation may be performed only to a limited extent.

Reconstructed epidermis versus human and animal skin in skin absorption studies.

European chemical policy in general and the REACH initiative in particular will increase the number of chemical substances submitted to toxicological evaluation by several orders of magnitude compared to the current status. To limit animal exposure the resulting enormous increase in testing, however, asks for validated in vitro test systems. While the OECD favours in vitro testing for cutaneous absorption using viable human and animal skin (Guideline 428) the availability of viable human skin is already limited today. We present a comparison of various in vitro techniques suitable for routine skin absorption studies including commercially available reconstructed human epidermis which may be a reliable alternative to excised human and animal skin. In order to develop a protocol for the subsequent transfer to partner laboratories the experimental set-up was analysed stepwise using the OECD reference compounds caffeine and testosterone. Franz cell type, the donor and receptor media for hydrophilic/lipophilic substances, albumin and tensid addition, and storage conditions of the excised skins were systematically varied. A protocol has been developed which now allows to proceed to the pre-validation process.

Regulatory use of (Q)SARs in toxicological hazard assessment strategies.

In 2001, the European Commission published a policy statement ("White Paper") on future chemicals regulation and risk reduction that proposed the use of non-animal test systems and tailor-made testing approaches, including (Q)SARs, to reduce financial costs and the number of test animals employed. The authors have compiled a database containing data submitted within the EU chemicals notification procedure. From these data, (Q)SARs for the prediction of local irritation/corrosion and/or sensitisation potential were developed and published. These (Q)SARs, together with an expert system supporting their use, will be submitted for official validation and application within regulatory hazard assessment strategies. The main features are: two sets of structural alerts for the prediction of skin sensitisation hazard classification as defined by the European risk phrase R43, comprising 15 rules for chemical substructures deemed to be sensitising by direct action with cells or proteins, and three rules for substructures acting indirectly, i.e., requiring biochemical transformation; a decision support system (DSS) for the prediction of skin and/or eye lesion potential built from information extracted from our database. This DSS combines SARs defining reactive chemical substructures relevant for local lesions to be classified, and QSARs for the prediction of the absence of such a potential. The role of the BfR database, and (Q)SARs derived from it, in the use of current and future (EU) testing strategies for irritation and sensitisation is discussed.

Permanent embryonic germ cell lines of BALB/cJ mice--an in vitro alternative for in vivo germ cell mutagenicity tests.

To offer a sensitive and predictive in vitro method to assess germ cell mutagenicity, we established primordial germ (PG) cell-derived permanent female and male embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of EG cells and differentiated fibroblast cells of the mouse cell line 3T3 to genotoxicants were tested comparatively under identical test conditions. Cytotoxicity assay was measured by the MTT test and genotoxic effects were determined by sister chromatid exchanges (SCE) rates induced by standard reference mutagens. Both methods are used to assign the chemicals to two classes of in vivo reproductive toxicity, non- and strongly genotoxic to germ cells. Applying linear discriminant analysis, a biostatistical prediction model (PM) was developed for the female cell line EG(3). This procedure identified a single variable, the Ig(SCE(200)EG(3)) as the statistically significant concentration related increase of 200% in the mean number of SCEs per metaphase spread after 3 h of exposure to be sufficient for separation into the classes: non- and strongly genotoxic to germ cells. Applying this PM to the training set of five genotoxic and three non-genotoxic test chemicals, 100% correct classifications were obtained.

Lessons learned from validation of in vitro toxicity test: from failure to acceptance into regulatory practice.

As no scientific approach or regulatory guidelines existed for the experimental validation of in vitro toxicity tests, in 1990 a US/European validation workshop agreed in Amden (Switzerland) on a simple definition of the validation process. Several international validation studies failed, although they were conducted according to these recommendations. Taking into account the lessons learned from this experience, a second validation workshop was held by ECVAM in Amden in 1994 to develop a more precisely defined validation concept. Prevalidation and the development of biostatistically defined prediction models were added as essential elements to the validation process. In 1995/1996 the ECVAM validation procedure was officially accepted by EU member countries and at the international level by the US regulatory agencies and the OECD. The improved validation concept was immediately introduced into ongoing validation studies. In 1996 the ECVAM/COLIPA validation study of the in vitro phototoxicity test, which was conducted according to the ECVAM/OECD validation concept, was finished successfully and in 1998 a supporting study on UV-filter chemicals was undertaken. In 1998 the 3T3 NRU PT in vitro phototoxicity test was the first experimentally validated in vitro toxicity test that was recommended for regulatory purposes by ESAC, the ECVAM Scientific Advisory Committee, and by the DG ENV of the EU Commission. Meanwhile, two in vitro skin corrosivity tests have successfully been validated by ECVAM. Finally, in June 2000 the three experimentally validated tests were accepted by EU member states for regulatory purposes as the first in vitro toxicity tests. In addition, ECVAM has funded a successful validation study of three in vitro embryotoxicity tests, which was conducted in 12 European laboratories and finished in July 2000. The three tests validated in this study were the whole embryo culture (WEC) test applied to rat embryos, the micromass (MM) test employing primary cultures of dissociated mouse limb bud cells and the mouse embryonic stem cell test (EST). Examples will be given of successful validation studies during the past decade with particular reference to in vitro toxicity tests that were evaluated for regulatory purposes either by the US validation centre ICCVAM or ECVAM in the fields of sensitisation, phototoxicity and embryotoxicity

[Report on the ICCVAM workshop on in vitro methods for assessing acute systemic toxicity]. / Ergebnisse des ICCVAM Workshop über in vitro Methoden zur Bewertung der akuten systemischen Toxizität.

It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cytotoxicity data to predict the in vivo starting doses, should be implemented into acute toxicity testing as soon as possible. The validity of the in vitro cytotoxicity data to establish appropriate starting doses for acute toxicity testing will be assessed experimentally. Secondly, in order to replace the use of animals in acute lethality testing a formal validation will be conducted in which the ability to predict rodent LD50 values and toxicity classes from cytotoxicity data will be evaluated.

[Permanent embryonic mouse germ cell-lines, an in vitro alternative to in vivo germ cell mutagenicity tests]. / Permanente embryonale Keimzelllinien - eine in vitro Alternative zur Beurteilung von Keimzellmutagenität.

Germ cell mutagenesis is required by the 7th amendment of the directive 67/548 EEC into the national regulations on existing chemicals. Officially accepted in vivo test systems for stage specific mutagenicity are the dominant lethal (DL) test and the specific locus test (SLT) in mice. An acceptable in vitro alternative designed to address germ cell mutagenesis and discriminate between male and female specific effects is not available at present. In order to offer a sensitive and predictive in vitro method to assess the genotoxic potential of chemical agents on male and female reproduction, we established primordial germ (PG) cell-derived permanent embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of the EG(3) cell line and differentiated fibroblast cells 3T3 were comparatively tested with cytotoxicity assay (MTT test ) and genotoxic studies (SCE-assay) under identical test conditions. The concentration-response curves reflected the female cell line EG(3) to be extremely sensitive concerning cytotoxic and genotoxic endpoints. Therefore this cell line was used to classify in vivo genotoxic and non-genotoxic test substances with different potential endpoints. Applying linear discriminant analysis three endpoints were identified for the correct classification (100%) of all test chemicals, namely the SCE(200) value (increase of 200% in the mean number of SCEs per metaphase spread) for EG(3) (3 hrs and 24 hrs assay) and the IC(5)0 value for EG(3) after 3 hrs of exposure to test chemicals.

[Prediction of human lethal concentrations by cytotoxicity data from 50 MEIC chemicals]. / Zur Prädiktion der letalen Konzentration beim Menschen mit Hilfe von Zytotoxitätsdaten von 50 MEIC-Stoffen.

Procedures for predicting human toxicity on the basis of cytotoxicity data for different chemicals are of current interest. The study was designed to clarify the possibility of predicting human toxicity by using the cytotoxicity values IC(50x) of the 50 MEIC chemicals listed in the Registry of Cytotoxicity (RC). All calculations with the data of cytotoxicity and in vivo toxicity were carried out uniformly by using standardised methods with the aim of comparing the results in the literature with each other. The following results were achieved: Firstly, the IC(50x) values in the RC are suited better for predicting human toxicity than IC(50) values determined in cell culture experiments with one cell type and one cytotoxic endpoint. This result is correct for the values of linear regression parameters as well as for the values of the prediction error (PE) defined by Ponsoda et al. (1997). Secondly, by using the geometrical mean of four lethal concentrations (LCx) - calculated from the tabulated lethal concentration (LC), lethal plasma concentration (LPC), clinical lethal concentration (CLC) and forensic lethal concentration (FLC) - the parameters for predicting the human toxicity were slightly improved. Moreover, these comparative examinations demonstrate that in all cases the cytotoxicity data are suited better for predicting acute animal toxicity than for predicting acute human toxicity. The results confirm, once more, the reliability and general validity of RC cytotoxicity data for examinations of different problems in cytotoxicology.

The ECVAM Prevalidation Study on the Use of EpiDerm for Skin Corrosivity Testing.

In 1996 and 1997, ECVAM supported a formal validation study on in vitro methods for predicting skin corrosivity. Two of the in vitro tests included in the study employed human skin models, the Skin2™ ZK1350 and EPISKIN™ models. In the ECVAM validation study, BASF, Huntingdon Life Sciences (HLS) and ZEBET tested the Skin2 human skin model, production of which ceased in October 1996, while the validation study was still in progress. Since both of the skin models had shown basic usefulness for corrosivity testing and, in particular, the EPISKIN corrosivity test had proved to be a scientifically valid test, the three laboratories decided to conduct a study to determine whether another commercially available human skin model, EpiDerm™, could also be successfully used to predict skin corrosivity. The study was performed according to the ECVAM prevalidation scheme, to allow for refinement of the test protocol and the prediction model, as well as for independent assessment of the performance of the refined methodology in a final blind trial in the three laboratories. In phase I of the study, ZEBET (Laboratory 1) drafted a Standard Operating Procedure (SOP), including a prediction model (PM1), and the project plan for the study. It was a major task to simplify an existing EpiDerm test protocol, which used the time-course of cytotoxicity as its endpoint. To evaluate the predictivity of the simplified method, which used only a 3-minute exposure to test chemicals, 50 chemicals representing a wide spectrum of chemical entities were tested, revealing that the test sensitivity was too low (65%), whereas the specificity was very high (88%). In addition, acceptance criteria for the negative and positive controls were established. Before proceeding to the next phase of the study, ZEBET distributed a refined SOP, data-recording software and documentation sheets, which allowed Good Laboratory Practice (GLP)-compliant quality assurance for each assay. The main goal of phase II was to produce sufficient data to assess the reproducibility of the EpiDerm skin corrosivity test after transfer to Laboratory 2 (HLS). Repeated testing of several chemicals in both laboratories revealed excellent intralaboratory and interlaboratory reproducibility. In addition, chemicals classified as "non-corrosive" (NC) with a 3-minute exposure in phase I, were re-tested by ZEBET with extended exposure periods of 1 hour and 4 hours. The test sensitivity could be significantly increased, if chemicals classified NC with a 3-minute exposure were tested with a 1-hour exposure. Before proceeding to the final blind trial, a refined SOP was drafted, according to which all chemicals had to be tested with exposure times of 3 minutes and 1 hour, and data for these two exposure times were used in the refined hierarchical prediction model, PM2. In phase III, the blind trial, BASF (Laboratory 3) joined the study. ECVAM selected 24 chemicals from the test chemical set used in the ECVAM skin corrosivity validation study, and BIBRA International (UK) purchased, coded and distributed the chemicals. Each chemical was tested twice, independently, according to the principles of GLP, and coded data were submitted to the Humboldt University (Berlin, Germany) for biostatistical analysis. The analysis revealed that the final test protocol and the refined prediction model (PM2) provided a highly balanced prediction of 88% sensitivity and 86% specificity, which is regarded as the best predictivity an in vitro skin corrosivity test can be expected to achieve. In conclusion, the EpiDerm skin corrosivity test gives an excellent prediction for a wide spectrum of chemicals, and could be used within the context of the new Annex V (EU Dangerous Substances Directive) test method (human skin model assay) for skin corrosion. The results obtained were reproducible, both within and between laboratories, and showed that EpiDerm could be used for testing a wide range of chemicals (both liquids and solids), including organic acids and bases, neutral organics, inorganic acids and bases, electrophiles and phenols. The concordances between the skin corrosivity classifications derived from the in vitro data were very good, and the test was able to distinguish.

Toxicity testing of polymer materials for dialysis equipment: reconsidering in vivo testing.

In fulfilment of the aims of the European Union Biocidal Directive (Directive 98/8/EC), Technical Guidance Documents are currently being compiled. Part I of these Technical Guidance Documents covers data requirements for active substances and biocidal products. The Three Rs principle has been applied in certain parts of the toxicity and ecotoxicity testing scheme for pesticides, such as testing for acute oral toxicity, skin and eye irritation, skin sensitisation, and dermal absorption. Further recommendations on how to proceed with regard to the continuing replacement, reduction and refinement of animal experiments in this field of regulatory testing are included for consideration. In this context, besides stressing the necessity to validate and accept further alternatives, emphasis is placed on providing the possibility of waiving unnecessary tests and on the continuous evaluation of whether certain tests are needed at all.

Eye Irritation Testing: The Way Forward. The Report and Recommendations of ECVAM Workshop 34.

This is the report of the thirty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). The workshop on Eye Irritation Testing: The Way Forward was held in Egham, UK, on 15-17 June 1998, under the chairmanship of Michael Balls (ECVAM, Italy). The workshop had two aims, the first of which was to review some of the previous multi-laboratory validation studies on alternatives to the Draize eye test and assess why many promising alternative methods were not successful in these studies. The second aim was to discuss strategies for making progress toward the short-term reduction, refinement, and eventual replacement, of the Draize test, including: a new approach to the validation of in vitro tests for eye irritancy, based on the use of reference standards, which promises to overcome some of the problems encountered in previous studies; the use of stepwise testing strategies which reduce and refine the use of animals in eye irritation testing; the use of multivariate and other statistical techniques for the further analysis of data generated in previous validation studies; and a programme of research aimed at understanding the underlying mechanisms of eye irritation.