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.

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.

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.

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.

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.

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.

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

Modern biostatistical methods for assessing in vitro/in vivo correlation of severely eye irritating chemicals in a validation study of in vitro alternatives to the Draize eye test.

The HET-CAM test and 3T3 cell neutral red uptake (NRU) cytotoxicity assay were evaluated in a national German validation project to replace the Draize eye test for classifying severely eye irritating chemicals, which have to be labelled 'R-41' according to EU regulations. As testing of 200 chemicals in the two in vitro assays did not sufficiently allow severely eye irritating chemicals to be identified and since the scoring system of the HET-CAM assay has been derived empirically, it was investigated whether modern biostatistical methods, for example discriminant analysis, would improve the selection of predictive endpoints of the HET-CAM assay. Comparison of HET-CAM data with adverse reactions observed in different tissues of the rabbit's eye proved that complex regression models are better describing in vitro /in vivo correlations than simple linear models. Discriminant analysis revealed that among the nine endpoints routinely determined in the HET-CAM test, coagulation was the only acceptable endpoint to classify severely irritating chemicals 'R-41' according to EU regulations. To identify R-41 chemicals the reaction time of appearance of coagulation of a 10% solution was the best discriminating factor and coagulation of the undiluted chemical for the less water-soluble ones. The results suggest that only R-41 chemicals are inducing coagulation of the CAM within 50 sec, and can therefore be classified without further testing in vivo. Stepwise discriminant analysis allowed an in vitro testing strategy to be developed to identify R-41 chemicals by combining coagulation data of the HET-CAM assay with cytotoxicity data. Validity of the model for future data sets was assessed by cross-validation. The results obtained with 200 chemicals under blind conditions suggest that this approach will provide an acceptable sensitivity, predictivity and percentage of false positive data for severely eye irritating chemicals.

application of the human dermal model skin(2) ZK 1350 to phototoxicity and skin corrosivity testing.

The human three-dimensional in vitro model Skin(2) ZK 1350(TM) [Advanced Tissue Sciences (ATS), La Jolla, USA] was tested for two in vitro toxicology applications, prediction of phototoxicity and classification of skin corrosivity. For phototoxicity testing chemicals were applied topically for 1 or 24 hr followed by 30 min of irradiation with a non-irritating dose of UVA. Phototoxicity was assessed 24 hr later by comparing cytotoxicity of UVA-exposed and non-exposed tissue in the MTT assay. In a European EC/COLIPA in vitro phototoxicity validation trial with 20 test chemicals (11 phototoxic and nine non-phototoxic), the best result was obtained with the Skin(2) ZK 1350 assay in the 24-hr exposure protocol where nine of the 11 phototoxins were classified correctly both at ATS and ZEBET. 6-Methylcoumarin could only be identified as a phototoxin when applied through the medium to the dermis side of the skin model for 24 hr. All of the nine non-phototoxic chemicals were identified correctly with either 1- or 24-hr preincubation. To classify chemicals as corrosive to the skin with an in vitro assay, ZEBET and two other laboratories tested 50 chemicals under blind conditions in the Skin(2) ZK 1350 model within a European ECVAM validation trial. The results obtained with the ZK 1350 assay showed a satisfactory classification of skin corrosive/non-corrosive chemicals and a sufficient prediction of the three UN Packing Groups for corrosive chemicals. Predictions obtained at ZEBET with the Skin(2) ZK 1350 model (sensitivity: 64%, specificity: 76%; positive and negative predictive values: 68%) compared with a sensitivity of 81% and a specificity of 77% as mean values for the three laboratories testing the model.

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.

Validation study of alternatives to the Draize eye irritation test in Germany: Cytotoxicity testing and HET-CAM test with 136 industrial chemicals.

According to OECD guideline 405 revised in 1987 Draize eye tests need not be performed for severely irritating and corrosive chemicals if results from 'well-validated alternative studies' are presented. In 1988 a validation study on alternatives to the Draize eye test was started in Germany to establish 'well-validated alternative methods' for this purpose. During database development, the last stage of the validation programme, 136 chemicals from the German chemical industry were classified in a blind trial with the 3T3 cell neutral red/kenacid blue cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test using fertile chicken eggs. The major goal of this stage of validation was to demonstrate the feasibility and limitations of the two alternative methods. Chemicals were, therefore, selected as representatives of chemical structural groups as well as of physicochemical and toxicological properties. In addition, some of the chemicals were chosen because they were of interest to the cosmetic and detergent industries. Draize eye testing data in vivo were provided by industry. In contrast to data from a previous interlaboratory assessment trial, it was impossible to correlate cytotoxicity data to the EEC classification for in vivo eye irritation. However, seven of 10 severely irritating chemicals (EEC labelling R-41) could be identified correctly in the HET-CAM assay, whereas test conditions of the study described here did not allow identification of irritating chemicals (EEC labelling R-36). The HET-CAM test is, therefore, fulfilling the criteria of a 'well-validated alternative method' according to OECD guideline 405 and should be incorporated into eye irritation testing at the earliest possible stage to reduce effectively the suffering of rabbits in the Draize eye test. Although an 80% correct prediction of 'non-labelled' chemicals in the HET-CAM test is encouraging, for safety assessment of non-irritant chemicals, for use as cosmetic formulations, for example, both government and industry will accept an in vitro assay only if its prediction of the absence of irritant properties is 100% correct.

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluation by the Management Team.

As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX(TM), the Skin(2TM) ZK1350 corrosivity test and EPISKIN(TM). Each test was conducted in three independent laboratories. 60 coded chemicals were tested. All of the tests evaluated showed acceptable intralaboratory and interlaboratory reproducibilities, and the TER, Skin(2) and EPISKIN tests proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols and soaps/surfactants. Two of the four tests evaluated, the TER assay and EPISKIN, met the criteria agreed by the Management Team concerning acceptable underprediction and overprediction rates for them to be considered scientifically validated for use as replacements for the animal test for distinguishing between corrosive and non-corrosive chemicals for all of the chemical types studied [objective (a)]. EPISKIN was the only test able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals, for all of the chemical types included, on an acceptable number of occasions [objective (b)]. The corrosive potentials of about 40% of the test chemicals could not be assessed with CORROSITEX, and the assay did not meet all of the criteria for it to be considered acceptable as a replacement test. However, CORROSITEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. The Skin(2) assay did not meet the criteria for it to be considered scientifically validated. Thus, the validities of (i) the TER and EPISKIN assays for discriminating corrosives from non-corrosives, and (ii) the EPISKIN assay for identifying correctly known R35/I and R34/II & III chemicals, have been demonstrated in this study. CORROSITEX appears to be valid when used only with certain types of chemicals.

EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell phototoxicity assay.

In a joint validation project eight laboratories from the European Cosmetic Industry Association (COLIPA) as well as FRAME (England) and ZEBET (Germany) are trying to develop validated in vitro methods to be incorporated into new international guidelines for acute phototoxicity testing. The first stage of the study involved selection of the most promising in vitro phototoxicity tests for further validation. 20 chemicals with known phototoxic properties (12 phototoxins, four UV-absorbing non-phototoxins and four non-UV absorbing non-phototoxins) were tested under identical conditions of UV exposure conditions (sun simulator, UVA 5 J/cm(2)) in a standardized cytotoxicity assay with Balb/c 3T3 fibroblasts (endpoint: neutral red uptake, NRU). 19 of the 20 chemicals were correctly classified by the 3T3 NRU phototoxicity test, and therefore, this simple assay for phototoxicity seems very promising and should be validated further.

The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test.

To date, no standardized international guideline for the testing of chemicals for phototoxic potential has been accepted for regulatory purposes. In 1991, the European Commission (EC), represented initially by the Directorate General XI and later by ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to establish a joint EU/COLIPA programme on the development and validation of in vitro phototoxicity tests. The first phase (phase I, 1992-93) was designed as a prevalidation study, to identify in vitro test procedures and test protocols for a formal validation trial under blind conditions. In the second phase (phase II, 1994-95), the formal validation study, the most promising in vitro phototoxicity tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. The 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) was performed as a core test in nine laboratories, since it provided the best results in phase I of the study. The purpose of phase II was to confirm the reliability and relevance of the in vitro tests for predicting phototoxic effects and for identifying phototoxic chemicals. In phase II the phototoxic potential of test chemicals in the 3T3 NRU PT test was either assessed by determining the phototoxicity factor (PIF) by using a cut-off value of 5 as in phase I of the study, or by determining the mean photo effect (MPE) by using a cut-off value of 0.1, as recently proposed by Holzhütter (1997). Results obtained with both approaches in the 3T3 NRU PT test in phase II were reproducible in the nine laboratories, and the correlation between in vitro and in vivo data was very high. Therefore, ECVAM and COLIPA conclude from this formal validation trial under blind conditions that the 3T3 NRU PT test is a scientifically validated in vitro test which is ready to be considered for regulatory purposes for assessing the phototoxic potential of chemicals. A draft OECD Guideline for "In Vitro Phototoxicity Testing", incorporating the standard protocol of the 3T3 NRU PT test, will be submitted to the OECD test guidelines programme in due course.

IRAG working group 2. CAM-based assays. Interagency Regulatory Alternatives Group.

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9-133 test chemicals, usually from the sponsor's product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the sigma MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearson's linear coefficient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols.

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.

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.

A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test.

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1µ/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100µ/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1µg/ml and 10µg/ml, and that false positive results can be obtained at concentrations greater than 100µg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100µg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.