Comparative proteomic profiling of the ovine and human PBMC inflammatory response.

Understanding the cellular and molecular mechanisms of inflammation requires robust animal models. Sheep are commonly used in immune-related studies, yet the validity of sheep as animal models for immune and inflammatory diseases remains to be established. This cross-species comparative study analyzed the in vitro inflammatory response of ovine (oPBMCs) and human PBMCs (hPBMCs) using mass spectrometry, profiling the proteome of the secretome and whole cell lysate. Of the entire cell lysate proteome (oPBMCs: 4217, hPBMCs: 4574 proteins) 47.8% and in the secretome proteome (oPBMCs: 1913, hPBMCs: 1375 proteins) 32.8% were orthologous between species, among them 32 orthologous CD antigens, indicating the presence of six immune cell subsets. Following inflammatory stimulation, 71 proteins in oPBMCs and 176 in hPBMCs showed differential abundance, with only 7 overlapping. Network and Gene Ontology analyses identified 16 shared inflammatory-related terms and 17 canonical pathways with similar activation/inhibition patterns in both species, demonstrating significant conservation in specific immune and inflammatory responses. However, ovine PMBCs also contained a unique WC1+γδ T-cell subset, not detected in hPBMCs. Furthermore, differences in the activation/inhibition trends of seven canonical pathways and the sets of DAPs between sheep and humans, emphasize the need to consider interspecies differences in translational studies and inflammation research.

Chemical substances and contact allergy--244 substances ranked according to allergenic potency.

From 1985 to 2001 a group consisting of thirty experts including dermatologists from universities, representatives from the chemical industry and from regulatory authorities elaborated and consequently decided on the potency ranking of chemicals with contact allergenic properties. These chemicals were defined either as synthetic chemicals or as chemicals identified as ingredients in natural products. On 244 substances clinical and experimental data on humans and results of animal tests as documented in the scientific literature were carefully collected and evaluated. This careful evaluation and assessment of these chemicals clearly demonstrate that ranking of substances according to their allergenic potency is possible and justified. It was decided to rank the most potent contact allergens in Category A of substances having significant allergenic properties. Substances with a solid-based indication of a contact allergenic potential and substances with the capacity of cross-reactions were listed in Category B and substances with insignificant or questionable allergenic effects were listed in Category C. An assessment of these compiled data is published here. Three Appendices give a comprehensive overview of the 98 substances listed in Category A, the 77 substances listed in Category B and the 69 substances listed in Category C.

A national validation project of alternative methods to the Draize rabbit eye test.

In June 1988, a 2.5-yr inter-laboratory study involving 13 toxicology laboratories was started in West Germany to validate alternative methods to the Draize rabbit eye test. The aim of this collaborative study is to validate the classification of chemicals with regard to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg test-chorioallantoic membrane (HET-CAM) assay. The results should make it possible to decide whether and to what extent the NR/KB cytotoxicity test and the HET-CAM assay can replace the Draize test. After two test trials, standard testing procedures and protocols were agreed on. In addition, to facilitate management of the data and to reduce costs, personal computer (PC) software was developed for both tests, which allows storage of all data on floppy discs and statistical analysis on PCs. During the preliminary phase, the applicability of the software was tested and corrected according to the experimental conditions of the validation study. Tests on the following chemicals have so far been completed, and reproducibility and repeatability have been determined: sodium dodecyl sulphate, triethanolamine, zinc pyridinethione, dimethylsulphoxide and butoxyethanol. Only zinc pyridinethione, which is severely irritating in vivo, could not be tested in the HET-CAM test. The preliminary phase has shown that the number of chemicals that can be tested in the HET-CAM test during the validation project will be limited by costs and management problems of manpower and time.

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. 1. Selection and Distribution of the Test Chemicals.

An international validation study on in vitro tests for skin corrosivity was conducted during 1996 and 1997 under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The main objectives of the study were to assess the performances of selected in vitro tests in discriminating between: (a) corrosives (C) and non-corrosives (NC), for selected groups of chemicals (e.g. organic acids, phenols) and/or for all chemicals (single chemical entities only); and (b) known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. Each test was evaluated for reliability and relevance by using a test set of 60 coded chemicals. In this paper, the test chemicals used in the validation study are identified; they include organic acids (6C/5NC), organic bases (7C/3NC), neutral organics (9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganic salts (1C/2NC), electrophiles (3C/5NC) and soaps/surfactants (3NC). The in vivo classifications and important physicochemical properties (e.g. logP, pKa) of the test chemicals are given. The main criterion for including chemicals in the test set was that their corrosivity classifications were based on unequivocal animal data. Where available, structure-activity information was also used to support the corrosivity classifications. Despite the small numbers of chemicals in some of the categories, it was felt that the test set chosen represented the best possible for evaluating the performances of the in vitro tests for predicting skin corrosivity, given the limited availability of unequivocal animal data. The prediction of skin corrosivity from pH data was also investigated for those chemicals with extreme pH values (i.e. pH2 or 11.5). Nine of the 12 strongly acidic or alkaline chemicals in the test set, which were predicted to be C on the basis of their pH values, had also been found to be C in vivo.

Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany.

A national interlaboratory study to validate two alternative methods to the Draize rabbit's eye test, co-ordinated by ZEBET at the German Federal Health Office (BGA), is described. The aim of the study is to classify chemicals according to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test. During the last two years 12 toxicology laboratories from industry, universities and other research institutions have tested 32 substances from a variety of chemical classes, characterized by a broad spectrum of locally irritating properties, using the NR/KB cytotoxicity test and the HET-CAM assay. Intra- and interlaboratory reproducibility of the two methods was investigated under standardized conditions. The so-far limited evaluation of the interlaboratory assessment phase of validation indicates that the results of the Draize rabbit's eye test correlate better with the results of the HET-CAM test than with those of the cytotoxicity test as far as false negative results are concerned. However, the intra- and interlaboratory reproducibility of the cytoxicity test is better than that of the HET-CAM test. The validation project has recently entered the stage of database development during which 150 chemicals will be tested in seven laboratories to provide information on whether and to what extent the NR/KB test and the HET-CAM test can replace the Draize rabbit's eye test for the classification and labelling of chemicals with regard to their eye irritation potential.

Evaluation of eye irritation potential: statistical analysis and tier testing strategies.

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

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.

Local irritation/corrosion testing strategies: extending a decision support system by applying self-learning classifiers.

Procedures have been established and tested for the extension of a decision support system (DSS) for the prediction of the local irritation/corrosion potential of chemicals by using self-learning classifiers. The different approaches (decision trees, distances examinations in a multidimensional space, k-nearest-neighbour method) have been implemented, tested and evaluated independently. A combination of all of the established extension approaches was also developed and tested. Self-learning classifiers are constructed "automatically" by a computer, i.e. they are not derived by a human expert, and thus they can be constructed with minimal effort. The classifiers presented here extend the existing DSS in a manner that increased significantly the predictive power of the extended system. However, automatically calculated results of self-learning classifiers are produced by a machine, and a machine is incapable of explaining the toxicological relevance of the results obtained. Thus, these results must be accepted, despite an inability to prove their reliability. Only the mathematical correctness of the method and the prediction rates for suitable test cases can lend some credibility to predictions produced by a computer calculating on a self-learning basis. This may not be adequate for regulatory hazard assessment purposes.

A Computer-Based Structure-Activity Relationship Method for Predicting the Toxic Effects of Organic Chemicals from Onedimensional Representations of their Molecular Structures.

A computer-based method is presented for the analysis and interpretation of structural formulae characterising chemical molecules. This method was developed to enable a computer to identify substructures of chemical molecules that are relevant in the context of specific toxicological questions. The new computer-based structure-examination method was used to develop the "structure" parts of several electronic structure-activity relationship models (SAR models) for analysing and interpreting the structural formula of a chemical from its one dimensional representation, by applying recursive principles and identifying partial isomorphic graphs. The structure-examination method is designed as part of an open-endpoint procedure to be used in expert systems, and could be applied in the construction of SAR models for almost all toxicological endpoints. The system was satisfactorily tested by identifying substructures relevant to severely damaging effects on skin and eyes.

Development of a decision support system for the introduction of alternative methods into local irritancy/corrosivity testing strategies. Creation of fundamental rules for a decision support system.

The notification procedure of the European Union (EU) for new chemicals requires the application of protocols on physicochemical and toxicological tests for the evaluation of physicochemical properties and probable toxic effects of each notified substance. A computerised database was developed from data sets and toxicological test protocols relating to substance properties responsible for skin and eye irritation/corrosion. To develop specific structure-activity relationship (SAR) models and to find rules for a decision support system (DSS) to predict local irritation/corrosion, physical property data, chemical structure data and toxicological data for approximately 1300 chemicals, each having a purity of 95% or more, were evaluated. The evaluation demonstrated that the lipid solubility and aqueous solubility of a chemical are relevant to, or - in some cases - responsible for, the observed local effects of a substance on the skins and eyes of rabbits. The octanol/water partition coefficient and the measured value of the surface tension of a saturated aqueous solution of the substance give additional information that permits the definition of detailed SAR algorithms that use measured solubility values. Data on melting points and vapour pressure can be used to assess the intensity and duration of local contact with a chemical. Considerations relating to the reactivity of a pure chemical can be based on molecular weight and the nature of the heteroatoms present. With respect to local lesions produced following contact with the skin and eyes of rabbits, the data evaluation revealed that no general "local irritation/corrosion potential" of a chemical can be defined. A variety of mechanisms are responsible for the formation of local lesions on the skin or in the eyes: serious lesions are produced by mechanisms different from those that cause moderate irritation in these organs. In order to develop a DSS that uses the information extracted from the database, chemical main groups were categorised on the basis of their empirical formulae, and rules were defined of the type IF (physicochemical property) A, THEN not (toxic) effect B, based on correlations between specific local effects and measured physicochemical values. Other rules of the type IF substructure A, THEN effect B were developed based on correlations between specific local effects and the submitted structural formulae. Reactive chemical substructures relevant to the formation of local lesions and rules for the prediction of the absence of any skin irritation potential were identified. Proposals are made relating to the development of alternatives to eye irritation testing with rabbits.

Development of a decision support system for the introduction of alternative methods into local irritancy/corrosivity testing strategies. Development of a relational database.

For new chemical substances that are notified within the European Union, data sets have to be submitted to the National Competent Authorities. The data submitted have to demonstrate the physicochemical and toxic properties of the new chemical, such as solubility, partition coefficients and spectra, as well as acute toxic properties and the potential to cause local irritant or corrosive effects. In order to minimise testing for notification purposes (for example, animal testing), it is necessary to develop stepwise assessment procedures, including structure-activity considerations, alternative methods (for example, in vitro tests), and computerised structure-activity relationship (SAR) models. An electronic database was developed which contains physicochemical and toxicological data on approximately 1300 chemical substances. It is used for regulatory structure-property relationship (SPR) and SAR considerations, and for the development of rules for a decision support system (DSS) for the introduction of alternative methods into local irritancy/corrosivity testing strategies. The information stored in the database is derived from proprietary data, so it is not possible to publish the data directly. Therefore, the database is evaluated by regulators, and the information derived from the data is used for the development of scientific information about SARs. This information can be published, for example, by means of tables correlating measured physicochemical values and specific toxic effects caused by the measured chemical. This information is introduced to the public by means of a DSS that predicts local irritant/corrosive potential of a chemical by listing so called exception rules of the kind IF (physicochemical property) A THEN not (toxic) Effect B and so-called structural rules of the kind IF Substructure A THEN Effect B. These DSS rules "translate" proprietary data into scientific knowledge that can be published.

Local irritation/corrosion testing strategies: development of a decision support system for the introduction of alternative methods.

The notification procedure for new chemicals of the European Union (EU) requires protocols on physicochemical and toxicological tests for the evaluation of physicochemical properties and probable toxic effects of each notified substance. In order to reduce the amount of animal testing, alternative methods should be introduced into toxicity testing. Therefore, we have developed a rule-based decision support system (DSS) for the prediction of the local corrosive/irritant properties of new chemicals. To this end, data on more than 1000 substances were examined, which resulted in approximately 180 "exception-rules" of the kind IF (physicochemical property) A THEN not (toxic) Effect B. In addition, the structural formulae of the chemicals were analysed, which resulted in approximately 160 "structure-rules" of the kind IF Substructure A THEN Effect B. The DSS can predict (based on theoretical structure-activity relationships) whether a chemical produces: a) corrosive effects (i.e. no testing is necessary; b) might have corrosive effects (i.e. no animal testing, in vitro tests are suitable) ; and c) will produce no effects or only marginal effects (i.e. animal tests are necessary based on current EU legislation for hazard assessment purposes). In addition, the DSS provides reliable data for legal classification and labelling based on a specific result.

[Conjunctival nevi in Denmark 1960-1980. A 21-year follow-up]. / Konjunktivale naevi i Danmark 1960-1980. Enogtyve års followup.

The clinicopathological characteristics of 343 naevi of the conjunctiva were studied. A significant increase in the number of naevi excised per year was observed. This may have been caused by an increased exposure to actinic rays. An approximately even distribution was found between the three main locations: caruncle, limbal area and eye ball. Intrastromal naevi were excised at a higher median age than compound naevi, and the lowest median age at excision was for junction naevi, which is in accordance with the known histopathological nature of naevi. Recurrence occurred in nine patients (2.7%), and one had transformed into a malignant melanoma. Eight of the recurring naevi were located in the limbal area. Eight of the nine patients were women, suggesting hormonal factors as a possible cause. Recommendations for the handling of conjunctival naevi are given, based on the present findings and on previous reports.

Validation project of alternatives for the Draize eye test.

The Federal Health Office (BGA) has started in June 1988 a national interlaboratory study to validate alternatives to the Draize test. It is supported by grants of the Department of Research and Technology (BMFT) of West Germany. The aim of this collaborative study is to validate the classification of chemicals with regard to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg test-chorioallantoic membrane (HET-CAM) assay, according to Lüpke. Under the current research scheme, which is coordinated by the West German Public Health Office (BGA), 14 toxicology laboratories in the chemical industry, universities, the BGA, and other research institutions are to study 44 substances with a variety of chemical, biochemical, and toxicological characteristics. The validation test is intended to provide comparative data for the development of an alternative routine test scheme. The collaborative study is performed under routine laboratory conditions. The results should allow a decision on whether and to what extent the results of the NR/KB and the HET-CAM assay can replace the Draize test.