EU Classification Research Articles

Selection of test methods to be included in a testing strategy to predict acute oral toxicity: An approach based on statistical analysis of data collected in phase 1 of the ACuteTox project

More than 50 different in vitro and in silico methods assessing specific organ- and system-toxicity, such as haemato-, neuro-, nephro- and hepatotoxicity, as well as intestinal absorption, distribution and metabolism, have been used in the first phase of the ACuteTox project to test a common set of 57 chemicals. This paper describes the methods used for statistical evaluation of concentration–response data collected for each of the endpoint assays, and for the development of a testing strategy applicable for acute toxicity classification of chemicals based on the achieved results of the concentration–response analysis. A final list of in vitro test methods considered to be promising candidates for building blocks of the testing strategy is presented. Only these selected test methods were further investigated in the prevalidation phase of the project. The test methods were chosen according to their reproducibility and reliability and most importantly, according to their potential to classify chemicals into the official acute oral toxicity categories of the EU Classification, Labelling and Packaging (CLP) Regulation. The potential of the test methods to correctly classify the chemicals was assessed by Classification and Regression Trees (CART) analysis.

Methylene diphenyl diisocyanate (MDI): EU classification; impact of REACH; worker and consumer exposure – perception versus facts

Methylene diphenyl diisocyanate (MDI) is being used increasingly as an alternative resin in the composite wood industry for environmental, indoor air, production capacity or quality reasons. This paper gives an overview of the hazards, classification, regulations and exposure risks related to the use of MDI and MDI bonded wood products. Its aim is to provide factual scientific information to address some of the concerns regularly encountered when introducing MDI as a resin. Like all chemicals, MDI has its hazards and is regulated within existing regulatory frameworks like Globally Harmonized System (GHS) the European Regulation for the REACH, Evaluation, Authorization and Restriction of Chemicals (REACH) and Classification, Labelling & Packaging (CLP). It is important to understand the risks associated with chemicals in the workplace to raise awareness with workers. Understanding safety issues has equal value in identifying where to implement risk reduction measures so workers are protected, and in ensuring investments are economically feasible. The inhalation exposure risks of MDI are given based on an extensive dataset and compared with other agents present in the workplace, i.e. wood dust and formaldehyde. In addition, this paper addresses the risk for consumers for the use of MDI bonded composite wood products (CWPs). The conclusion is that MDI has been classified as a hazardous chemical; however, safe use can be demonstrated within the REACH framework. The exposure risks of MDI, compared to other chemical agents present in the workplace, are significantly lower and can be reduced further if actions are taken to control wood dust. Furthermore, there is no need for concern regarding exposure to consumers if MDI bonded CWPs are used in buildings or homes.

Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations

Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labelling and Packaging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.

Assessing the potency of respiratory allergens: Uncertainties and challenges

In contrast to skin sensitisation, there are no accepted tests for the identification of chemicals or proteins with the potential to cause sensitisation of the respiratory tract. Although progress has been made, the assessment of respiratory sensitisation potential remains associated with significant challenges and uncertainties. Nevertheless, there is interest in determining whether it is possible to assess the relative potency of respiratory sensitisers. The second Adaptation to Technical Progress (ATP) to the EU Classification, Labelling and Packaging (CLP) Regulation recently introduced changes to criteria for classification and labelling of chemicals and preparations, bringing it in line with the 3rd revision to the UN Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Among other things, the second ATP introduces sub-categories for respiratory and skin sensitisers, discriminating between strong sensitisers and other sensitisers. Here we examine whether such categorisation of protein and/or chemical respiratory allergens is realistic and/or feasible. For this purpose comparisons have been drawn with skin sensitisation, where potency categorisation has now been widely accepted and successfully integrated into the regulatory process. The conclusion drawn is that, on the basis of the currently available information, potency categorisation for respiratory sensitisers is premature and could potentially be misleading.

Environmental and health hazard ranking and assessment of plastic polymers based on chemical composition

Plastics constitute a large material group with a global annual production that has doubled in 15years (245million tonnes in 2008). Plastics are present everywhere in society and the environment, especially the marine environment, where large amounts of plastic waste accumulate. The knowledge of human and environmental hazards and risks from chemicals associated with the diversity of plastic products is very limited. Most chemicals used for producing plastic polymers are derived from non-renewable crude oil, and several are hazardous. These may be released during the production, use and disposal of the plastic product. In this study the environmental and health hazards of chemicals used in 55 thermoplastic and thermosetting polymers were identified and compiled. A hazard ranking model was developed for the hazard classes and categories in the EU classification and labelling (CLP) regulation which is based on the UN Globally Harmonized System. The polymers were ranked based on monomer hazard classifications, and initial assessments were made. The polymers that ranked as most hazardous are made of monomers classified as mutagenic and/or carcinogenic (category 1A or 1B). These belong to the polymer families of polyurethanes, polyacrylonitriles, polyvinyl chloride, epoxy resins, and styrenic copolymers. All have a large global annual production (1–37million tonnes). A considerable number of polymers (31 out of 55) are made of monomers that belong to the two worst of the ranking model's five hazard levels, i.e. levels IV–V. The polymers that are made of level IV monomers and have a large global annual production (1–5million tonnes) are phenol formaldehyde resins, unsaturated polyesters, polycarbonate, polymethyl methacrylate, and urea-formaldehyde resins. This study has identified hazardous substances used in polymer production for which the risks should be evaluated for decisions on the need for risk reduction measures, substitution, or even phase out.

The Panel Report on Tariff Treatment of Certain Information Technology Products: Classification Is the Key to Effective Implementation

The Report of the Panel in Tariff Treatment of Certain Information Technology Products confirms the wide scope of concession granted under the ITA in relation to set-top boxes with a communication function. The EU now needs to bring its provisions on classification in line with these commitments. The Report leaves open the question of when an ITA set-top box will be so altered by the development of hitherto ancillary functions that it no longer falls within the scope of the concession. This article sets out some views on the correct classification of set-top boxes with a communication function (STBCs) and with an HDD and explains why the contested CNEN should have been considered as incompatible with EU classification rules.

Eye irritation potential: Usefulness of the HET-CAM under the Globally Harmonized System of Classification and Labeling of Chemicals (GHS)

Extensive research has been conducted over the past decades to develop alternatives to the rabbit eye irritation test (Draize test) used in a regulatory context to assess eye irritation potentials. Although no single in vitro test has emerged as being completely acceptable for full replacement, various tests are considered to be suitable and are regularly used to assess certain aspects. Amongst these, the Hen’s Egg Test Chorioallantoic Membrane (HET-CAM) has gained regulatory acceptance in various countries to classify severe eye irritants. In this retrospective study, historical eye irritation data (in vivo and in vitro) from 137 samples (approx. 75% non-irritants; 25% (severe) irritants) tested both in the HET-CAM and Draize eye test was compared with regard to the predicted eye irritation classes under the GHS and the traditional EU classification system (DSD).The overall concordance was in the range of 80–90%. A high specificity (96–98%, depending on the classification system and the chosen discrimination) but rather low sensitivity (48–65%) was observed. The study indicates that HET-CAM results are useful as part of weight-of-evidence assessments or in tiered approaches to assess eye irritation potentials rather than as stand-alone classification method.

Evaluation of the Acute Oral Toxicity Class of Trinuclear Chromium(III) Glycinate Complex in Rat

Chromium(III) is considered as an essential element playing a role in carbohydrate and lipid metabolism, and various chemical forms of this element are widely used in dietary supplements. A new trinuclear chromium(III) glycinate complex [Cr3O(NH2CH2CO2)6(H2O)3]+NO3− (CrGly), an analogue of Cr3 (trinuclear Cr(III) propionate complex) has been synthesized as a potential source of supplementary Cr. In this study, we evaluated the acute toxicity class of CrGly in Wistar rats applying the OECD 423 procedure. Male and female Wistar rats (n = 12, 6 ♀ and 6 ♂) were given by gavage either a single dose of CrGly 2,000 mg/kg body mass (equals to 300 mg Cr(III)/kg body mass; in aqueous solution) or equivalent volumes of distilled water and fed ad libitum commercial Labofeed B diet, and observed carefully for 14 days, then sacrificed to collect blood and internal organs for biochemical and histologic examination. No death cases were detected. No abnormalities in animal behavior, body mass gains, gross organ histology, or blood morphology and biochemistry were observed. The results demonstrate that LD50 of CrGly is greater than 2,000 mg/kg when administrated orally to rat; thus, this compound appears to belong to the fifth category in the GHS system or the fourth class (“unclassified”) in the EU classification system.

Effects of the rearing season on carcass and meat quality of suckling Apennine light lambs

The aim of this study was to evaluate the effects of the rearing season (autumn vs winter) on the carcass and meat quality of light lambs, obtained according to the traditional farming system usual in central Italy. Eighty carcasses from 60 d+/-3 old unweaned Apennine single birth male lambs, permanently reared indoor, half in autumn (receiving milk from ewes permanently pastured) and half in winter (which dams did never acceded to pasture), were weighed, classified according to the EU classification system for light lambs, and their Longissimus lumborum meat was analysed for pH, colour, drip and cooking losses, proximate composition and fatty acids profile of intramuscular fat. Most of the carcasses fully responded to the 1st quality EU requirements and no effects of the rearing season was evident on carcass characteristics. On the contrary, L. lumborum of lambs born and reared in autumn, receiving milk from ewes permanently pastured, evidenced a lower Lightness L* (P=0.02), a higher Chroma C* (P=0.01), with a higher fat content (P=0.04) than lambs reared in winter, which dams were permanently stall-fed. Moreover the intramuscular fat of the former was characterized by a greater PUFA concentration (P=0.01), a more favourable n-6/n-3 ratio (P<0.001), and a higher CLA content (P<0.001) than the latter, as a result of the difference related to the sheep traditional feeding system.

An evaluation of a cultured human corneal epithelial tissue model for the determination of the ocular irritation potential of pharmaceutical process materials

Irritation and other forms of local toxicity following contact with eyes is a potentially serious problem arising from occupational exposure to chemicals. Traditionally, evaluation of the irritant potential of novel chemicals has relied on the use of in vivo studies with rabbits. Concerns about the predictive potential of in vivo methods for human hazard and demand for economical and rapid screening of chemicals has stimulated a great deal of work to investigate in vitro alternatives for evaluating ocular irritation potential. This publication describes a screening study to assess a reconstituted corneal epithelial culture system, as an alternative for testing for ocular irritation with pharmaceutical process materials, extending the chemical domain with which this system has been tested. A total of 21 test chemicals were applied to commercially supplied reconstituted human corneal epithelial (HCE) cultures and effects on tissue viability (MTT reduction assay), tissue histology and IL-alpha expression were assessed. Positive controls (0.5% and 1% SDS) showed dose- and time-related adverse effects on tissues, consistent with known irritant effects. Negative controls showed no histological changes and retained high viability throughout the time-course of the experiment. Concordance was excellent with accuracy at each sampling time point of over 80% when viability (MTT reduction) was compared with existing EU classification of the test articles for ocular irritation (classification based on results of in vivo evaluation). Tissue viability as estimated by MTT reduction appears most useful as the primary means of assessing the irritation potential of the chemicals. Histopathological examination generally agreed with the results of the MTT assay. However, the use of cytokine analysis will need further consideration as results for this parameter showed no relationship with known irritation potential. These results infer that HCE cultures, alone or as a part of a tiered hazard screening programme, have promise for use in reducing reliance on live subject tests and contribute to generation of an appropriate hazard classification and label advice.

Experience with the HET-CAM Method in the Routine Testing of a Broad Variety of Chemicals and Formulations

Data on eye irritation are generally needed for the hazard identification of chemicals. For the routine testing of a broad variety of chemicals and formulations, we have used the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method. In the course of a tiered-testing strategy, and due to the lack of global regulatory acceptance of the HET-CAM method, we have also performed the Rabbit Eye Irritation test according to OECD Test Guideline 405. Of the 145 substances tested, 76% were classified as non-irritant/mild irritant and 13% were identified as irritant in vivo, according to the EU classification system (61% and 28%, respectively, with the GHS classification). The remaining 11% were severe irritants in vivo, based on the irreversibility of the effects and not due to sufficiently high irritation scores in the three days following application. The retrospective analysis revealed that the overall accuracy of the HET-CAM assay was 65% and the overall rates of false-negatives (FN) and false-positives (FP) were 50% and 33%, respectively. The HET-CAM assay was sufficiently specific (few FP) for water-soluble substances, but failed to identify nearly all the severe irritants within this group. In contrast, it was highly sensitive (no FN) for non-soluble and oil-soluble substances, but the specificity for this group was rather low. Therefore, we conclude that the HET-CAM assay is not useful in our tiered-testing strategy for eye irritation testing. However, for water-insoluble substances, it might be applicable in combination with another in vitro method, provided that regulatory acceptance is gained.

Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data

Efforts to replace the rabbit skin irritation test have been underway for many years, encouraged by the EU Cosmetics Directive and REACH. Recently various in vitro tests have been developed, evaluated and validated. A key difficulty in confirming the validity of in vitro methods is that animal data are scarce and of limited utility for prediction of human effects, which adversely impacts their acceptance. This study examines whether in vivo or in vitro data most accurately predicted human effects. Using the 4-hr human patch test (HPT) we examined a number of chemicals whose EU classification of skin irritancy is known to be borderline, or where in vitro methods provided conflicting results. Of the 16 chemicals classified as irritants in the rabbit, only five substances were found to be significantly irritating to human skin. Concordance of the rabbit test with the 4-hr HPT was only 56%, whereas concordance of human epidermis models with human data was 76% (EpiDerm) and 70% (EPISKIN). The results confirm observations that rabbits overpredict skin effects in humans. Therefore, when validating in vitro methods, all available information, including human data, should be taken into account before making conclusions about their predictive capacity.

Evaluation of the acute oral toxicity class of tricentric chromium(III) propionate complex in rat

Chromium(III) is an essential element for carbohydrate and lipid metabolism, and various chemical forms of this element are widely used in dietary supplements. Of particular interest is [Cr3O(O2CCH2CH3)6(H2O)3]+ cation (CrProp), that has been proposed as an alternative source of Cr. However, its safety has not been studied completely. In this study, we evaluated the acute toxicity class of CrProp in Wistar rats applying the OECD 423 procedure. Male and female Wistar rats (n=12, 6 ♀ and 6 ♂) were given by gavage either a single dose of CrProp 2000mgkg−1 body mass or equivalent volumes of distilled water, and fed Labofeed B diet, and observed carefully for 14days, than sacrificed to collect samples for biochemical and histologic examination. No death cases were detected, no major abnormalities in animal behaviour, body mass gains, gross organ histology, and blood morphology, and biochemistry were observed, except some changes of liver mass and the activity of ALT in female rats. The results demonstrate that LD50 of CrProp is greater than 2000mgkg−1 when administrated orally to rat, thus this compound appears to be belong the fifth category in the GHS system or the fourth class (“unclassified”) in the EU classification system.

In Vitro Skin Irritation Testing: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol

A skin irritation test (SIT) utilising a common protocol for two in vitro reconstructed human epidermal (RhE) models, EPISKIN and EpiDerm, was developed, optimised and evaluated as a replacement for the in vivo rabbit skin irritation test in an ECVAM-sponsored validation study. In 2007, both RhE models were recognised by an independent peer-review panel and the ECVAM Scientific Advisory Committee (ESAC) as validated for use with the common SIT protocol. The EPISKIN SIT was endorsed as a full replacement of the in vivo rabbit test. Since the EpiDerm SIT proved to be less sensitive than the in vivo test and the EPISKIN SIT, the test was recognised as a validated component of a tiered testing strategy, in which positive results are accepted and negative results require further confirmation. The ESAC, in its April 2007 statement, also recommended increasing the sensitivity of the EpiDerm SIT, in order to gain the full acceptance. Analysis of the EpiDerm and EPISKIN data from the ECVAM validation study indicated that the lower sensitivity of the EpiDerm SIT might be linked to the more robust barrier properties of the EpiDerm model. This hypothesis was also in line with results published previously. To overcome the relatively low sensitivity of the EpiDerm protocol as a hindrance to full regulatory acceptance, a modification of exposure conditions was introduced into the protocol to achieve better agreement with the in vivo rabbit data. In the Modified EpiDerm SIT protocol, the test chemical exposure time was increased from 15 minutes to 60 minutes. In addition, part of the exposure was performed at 37 degrees C. When the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay endpoint was used for classification, a significant increase of sensitivity was obtained (86.1%), whilst maintaining the high specificity of the method (76.3%). With the change to the EU classification system, which now uses higher cut-off for the classification of irritants, the sensitivity of the Modified EpiDerm SIT increased to above 90%. The measurement of interleukin (IL)-1alpha release did not further contribute to improvement of the method. The results demonstrate that the modified EpiDerm SIT protocol has the required sensitivity and specificity to be accepted as a stand alone method for complete replacement of the in vivo rabbit test.

In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic™ HCE model: Application to 435 substances from consumer products industry

The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20 years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic™ HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic™ HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation.

The delineation of nanoscience and nanotechnology in terms of journals and patents: A most recent update

The journal set which provides a representation of nanoscience and nanotechnology at the interfaces among applied physics, chemistry, and the life sciences is developing rapidly because of the introduction of new journals. The relevant contributions of nations can be expected to change according to the representations of the relevant interfaces among journal sets. In the 2005 set the position of the USA decreased more than in the 2004-set, while the EU-27 gained in terms of its percentage of world share of citations. The tag “Y01N” which was newly added to the EU classification system for patents, allows for the visualization of national profiles of nanotechnology in terms of relevant patents and patent classes.

The ECVAM International Validation Study on in Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN, EpiDerm), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU classification scheme, "R38" and "no label" (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin-1alpha (IL-1alpha), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1alpha release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1alpha assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1alpha release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.

The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Selection of Test Chemicals

The ECVAM-funded skin irritation validation study (SIVS) was initiated in 2003, with the aim to evaluate whether the EpiDerm, EPISKIN and the SIFT alternative methods were able to reliably identify skin irritant and non-irritant chemicals, and could therefore be candidates for replacing the rabbit Draize test for skin irritation. The primary goal of the study was to evaluate the predictive capacity of the assays with regard to the EU classification system, which employs the risk phrases, "R38", for skin irritants, and "no label" for non-irritants. A secondary objective was the retrospective analysis of the data, to assess whether the in vitro tests would be able to discriminate between strong irritants (category 2), mild irritants (category 3) and non-irritants (no category), as defined by the OECD and United Nations proposal for a Globally Harmonised System (GHS) for the classification and labelling of dermal irritancy. A Chemicals Selection Sub-Committee (CSSC) was appointed to identify test chemicals to be used in the SIVS, for which existing, high quality in vivo data were available, with which to correlate the in vitro measurements. Since chemicals from the European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) database of reference chemicals for skin irritation/skin corrosion had been extensively used in preceding studies, the CSSC made use of novel sources for potential test chemicals. The first source of chemicals screened was the New Chemicals Database (NCD), which is the central archive within the EU notification scheme for 'new' commercial chemicals. Data registered in the NCD originate from standard assays, submitted in compliance with the legislation which regulates the marketing of industrial chemicals, and are subject to quality assurance by the competent authorities of the EU Member States. In addition, to obtain 'existing' chemicals which were readily available from major manufacturing and/or distribution sources, additional databases were surveyed, such as the Toxic Substance Control Act (TSCA) database maintained by the US Environmental Protection Agency (EPA), and the ECETOC database, with the exclusion of the chemicals used in the previous optimisation and prevalidation phases. A total of approximately 3500 chemicals from the NCD and 1600 from the additional databases were screened. Pre-determined selection criteria were applied, primarily to ensure the quality of the in vivo data and the practicability of their use in testing. Overall, the number of chemicals fulfilling the CSSC selection criteria was found to be limited, particularly in the case of GHS category 2 chemicals. However, a total set of 60 chemicals were selected and proposed to the Management Team of the SIVS for independent coding and supply to the participating laboratories. The selected chemicals: i) represented statistically justified sample sizes for distinguishing R38 from no-label chemicals; ii) provided a balanced representation of the three GHS categories, to allow for the post hoc evaluation of the performance of the assays for that classification system; and iii) acknowledged, to a certain degree, the large prevalence known to exist for chemicals which have oedema and erythema scores of 0. The selected chemicals represented a variety of molecular structures, functional chemical groups, and effect and use categories, as well as a wide range of physico-chemical properties. They represented a challenging set of chemicals, relevant to current industrial commerce, with which to validate the alternative methods.

An evaluation of selected valid and mechanistically based SARs for skin sensitisation

Skin sensitisation which clinically is manifested as allergic contact dermatitis is an important health endpoint to be taken into account in the risk assessment of chemicals. In the new EU chemical policy REACH, information on skin sensitisation potential will have to be provided for chemicals manufactured or imported equal to or above 1 tonne/year and it is estimated that the highest number of substances will need to be tested for skin sensitisation. Within REACH, the use of (Q)SARs and other alternative methods is strongly encouraged in order to reduce the number of animal tests. The goal of this project was to test the applicability of SARs and structural alerts for skin sensitisation in the REACH framework. SARs and structural alerts which were found in the literature were analysed and further refined by using a number of examples (substances) that had not been previously used in the derivation of the published SARs. For substances which fit within the boundaries of the proposed alerts, it is assumed that they would be classified as sensitising in accordance with EU classification and labelling rules.

Development of an in vitro corrosion/irritation prediction assay using the EpiDerm™ skin model

A validation of the in vitro skin corrosion method using the EpiDerm™ skin model was performed using 12 recommended chemicals. All chemicals were correctly classified by OECD test guideline 431. In order to predict corrosion and/or irritation potential, additional compound exposure times and IL-1α measurements were included in a tiered testing approach. Four exposure times were performed followed by MTT (viability) and IL-1α measurement. This allowed classification of corrosive chemicals (OECD guideline 431) and those likely to be severe irritants. If the chemical was found to be corrosive or a severe irritant, no further experimental work was performed, otherwise a second experiment was performed using three further exposure times (same endpoints). The second experiment provided information on whether the chemical was likely to be a moderate/mild irritant. If the chemical was negative following both experiments, it was predicted as non-corrosive/non-irritating. A total of 12 chemicals were tested in the irritation or combined assay (five non-irritants, seven irritants). Specificity (% non-irritants concurring with EU classification) was 60% (MTT) and 100% (MTT + IL-1α). Sensitivity (% irritants concurring with EU classification) was 86% (MTT) and 86% (MTT + IL-1α). Accuracy (% chemicals correctly identified) was 75% (MTT) and 92% (MTT + IL-1α).