ECVAM s Activities in Validating Alternative Tests for Skin Corrosion and Irritation

Transcription

1 ATLA 30, Supplement 2, 61 67, ECVAM s Activities in Validating Alternative Tests for Skin Corrosion and Irritation Julia H. Fentem 1 and Philip A. Botham 2 1Safety & Environmental Assurance Centre (SEAC), Unilever Colworth Laboratory, Sharnbrook, Bedfordshire MK44 1LQ, UK; 2 Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK Summary ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commerciallyproduced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study. Key words: alternative methods, human skin model, in vitro, prevalidation, skin, skin corrosion, skin irritation, validation. Introduction Considerable progress has been made in recent years in validating in vitro alternatives to animal tests. In February 2000, the European Union (EU) Member States approved the first replacement alternative (in vitro) methods to be mandated for use for regulatory toxicity testing. In vitro tests for skin corrosion (the rat skin transcutaneous electrical resistance [TER] method and tests employing human skin models [1, 2]) and phototoxicity (the 3T3 neutral red uptake [NRU] phototoxicity test [3, 4]), have both been shown unequivocally to be reliable and relevant, in extensive prevalidation and formal validation studies conducted under the auspices of ECVAM. During the past 7 years, ECVAM has been proactively involved in funding, managing and coordinating the independent evaluation, prevalidation and validation of in vitro alternatives for skin corrosion (1, 2, 5) and skin irritation (6 8). Significant progress has been made. The validation and regulatory acceptance of alternative test methods for skin corrosion, and the prevalidation of in vitro tests for skin irritation, are reviewed in this paper. Skin Corrosion The assessment of acute skin corrosion/irritation potential is included in international regulatory requirements for the testing of chemicals (9). The standard approach used involves applying the test material to the shaved skin of albino rabbits. Testing for skin corrosion in laboratory animals has the potential to cause them considerable discomfort or pain, and it is recognised that the response in the rabbit is not always predictive of that found in humans. For these reasons, considerable effort has been directed in recent years toward the development and evaluation of alternative test methods for predicting chemical-induced acute dermal corrosion and irritation (1, 2, 6 8, 10; Table 1). Skin corrosivity testing is a relatively simple procedure in biological terms (9). The endpoint is severe and irreversible tissue destruction, not a subtle biological change, and the application route is topical, with no problems of dilution or distribution. These two factors made the development of non-animal methods for the prediction of skin corrosion easier than for other toxic effects exerted by more-subtle, multifactorial, mechanisms. Nevertheless, the successful validation of in vitro tests for skin corrosion (Table 1) represents a significant achievement in relation to the replacement of toxicity tests known to

2 62 J.H. Fentem & P.A. Botham Table 1: Overview of the validation and regulatory acceptance status of in vitro methods for skin corrosion and skin irritation Validation Method Test system Endpoint Applicability authority Status References Skin corrosion Rat skin transcutaneous Excised rat skin Electrical resistance General; additional dye ECVAM Validated; accepted by regulatory Fentem et al. (1) electrical resistance binding step for authorities for skin corrosion ECVAM (11) (TER) assay surfactants and solvents testing in the EU; OECD TG431 EC (14) EPISKIN human skin Reconstructed Cell viability General; a few materials ECVAM Validated, accepted by regulatory Fentem et al. (1) model (commercial human epidermal (MTT reduction assay) may interfere with MTT authorites for skin corrosion ECVAM (11) system) equivalent reduction testing in the EU; OECD TG431 EC (14) EpiDerm human skin Reconstructed Cell viability General; a few materials ECVAM Validated; accepted by regulatory Liebsch et al. (2) model (commercial human epidermal (MTT reduction assay) may interfere with MTT authorities for skin corrosion ECVAM (13) system) equivalent reduction testing in the EU; OECD TG431 EC (14) CORROSITEX Reconstituted Colour or physical Mainly acids, bases and ICCVAM Validated and endorsed (US and Fentem et al. (1) (commercial system) collagen matrix change in indicator derivatives (ECVAM) EU) method for skin corrosion NIH (15) chemical detection testing of acids, bases and ECVAM (16) system derivatives Skin irritation EPISKIN human skin Reconstructed Cell viability General; a few materials ECVAM Protocol modification and Fentem et al. (6) model (commercial human epidermal (MTT reduction assay) may interfere with MTT prevalidation (validation study Zuang et al. (8) system) equivalent reduction under discussion) Portes et al. (18) EpiDerm human skin Reconstructed Cell viability General; a few materials ECVAM Protocol modification and Fentem et al. (6) model (commercial human epidermal (MTT reduction assay) may interfere with MTT prevalidation (validation study Zuang et al. (8) system) equivalent reduction under discussion) Pig ear test Pig ear Transepidermal water General ECVAM Further development Fentem et al. (6) loss (TEWL) Zuang et al. (8) Mouse skin integrity Excised mouse TEWL and electrical General; a few materials ECVAM Protocol modification and Fentem et al. (6) function test (SIFT) skin resistance (ER) may interfere with either prevalidation (validation study Zuang et al. (8) TEWL or ER determination under discussion)

3 Alternative tests for skin irritation 63 cause considerable animal pain due to the nature of the endpoint under evaluation. and was also published in the scientific literature (11). Prevalidation A prevalidation study on in vitro skin corrosivity testing was conducted during 1993 and 1994 (10), as a first step toward defining those alternative tests that could be used within the context of OECD testing guideline 404 (9). Three tests were included in the prevalidation study: a) the rat skin TER assay; b) CORROSITEX (In Vitro International, Irvine, USA); and c) the Skin 2 ZK1350 corrosivity test (Advanced Tissue Sciences, La Jolla, USA). Fifty coded chemicals (25 corrosives [C] and 25 noncorrosives [NC)]) were tested. The report on the outcome of the prevalidation study, which was published as an ECVAM workshop report (10), recommended that a formal validation study should be conducted on alternative methods for skin corrosivity testing. ECVAM validation study An international validation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity was conducted during 1996 and 1997 under the auspices of ECVAM (1). The main objectives of the study were to: a) identify tests capable of discriminating corrosives (C) from non-corrosives (NC) for selected types of chemicals and/or all chemicals; and b) determine whether these tests could correctly identify known R35 (UN packing group I) and R34 (UN packing groups II and III) chemicals. The tests evaluated were the rat skin TER assay, CORROSI- TEX, the Skin 2 ZK1350 corrosivity test, and EPISKIN (EPISKIN, Chaponost, France). Each test was conducted in three independent laboratories. Sixty coded chemicals were tested (1, 5). Two of the tests evaluated, the TER and EPISKIN assays, met the criteria agreed by the Management Team (MT) concerning acceptable reproducibility and predictive ability (1), for them to be considered scientifically validated for use as replacements for the rabbit test for distinguishing between C and NC chemicals for all of the chemical types studied (objective a). EPISKIN was also able to distinguish between known R35/UN packing group I and R34/UN packing groups II and III chemicals, for all of the chemical types included in the study, on an acceptable number of occasions (objective b). The scientific validity of the rat skin TER and EPISKIN tests was endorsed by the ECVAM Scientific Advisory Committee (ESAC) in March 1998, and a signed statement to this effect was issued jointly by ECVAM (DG Joint Research Centre) and DG Environment of the European Commission (EC). This was disseminated widely, Catch-up validation An ECVAM-funded prevalidation study on the EpiDerm (MatTek, Ashford, USA) skin corrosivity test was coordinated by ZEBET (BgVV, Germany) during 1997 and 1998 (2). Phase III of this study was considered to be a catch-up validation activity for a test protocol which was similar to one which had already been successful in a formal validation study (1). The objective of the study was to determine whether a test protocol developed for another human skin model (i.e. in addition to that for EPISKIN) could similarly discriminate C from NC for various chemical types. The test was conducted in three independent laboratories, according to the ECVAM prevalidation scheme (12). In phase III, 24 coded chemicals (12 C, 12 NC) were tested; these were independently selected to be representative of the set of 60 chemicals tested in the ECVAM validation study (1, 5). The results obtained were reproducible, both within and between the three laboratories. As had been found with the rat skin TER and EPISKIN assays, the EpiDerm test proved applicable to testing a diverse group of chemicals (both liquids and solids). The concordances between the skin corrosivity classifications derived from the in vitro data and from the in vivo data were very good, and the test was able to distinguish between C and NC chemicals for all of the chemical types studied (2). Following a critical review of the test protocol and of all data supporting the validity of the EpiDerm test for skin corrosion, the method was endorsed as scientifically valid by the ESAC in March Again, a signed statement to this effect was issued and circulated by the EC, and this was published (13). Regulatory acceptance Further to the endorsement of the scientific validity of the rat skin TER and EPISKIN assays by the ESAC, several EC services (DG Environment and DG Enterprise), and the Scientific Committee for Cosmetics and Non-Food Products (SCCNFP), reviewed all the relevant documentation and subsequently added their endorsements to the ESAC statements. A draft guideline on the use of the TER and human skin model assays for skin corrosion testing was prepared by the MT of the validation study, which was jointly submitted to the OECD Secretariat in December 1998 by DG Environment (on behalf of the EC) and the UK Government authorities. A draft Annex V test method on skin corrosion was also prepared in February 1999, for

4 64 J.H. Fentem & P.A. Botham discussion with the EU National Coordinators for Test Methods. On 4 February 2000, the EU Competent Authorities approved the draft Annex V test method on skin corrosion, which makes the use of validated in vitro tests for this endpoint mandatory in the EU Member States. In June 2000, test method B.40, Skin Corrosion was incorporated into the Dangerous Substances Directive by way of Directive 2000/33/EC of 25 April 2000, the 27th adaptation to technical progress of Directive 67/548/EEC (14). In November 2001, an OECD workshop involving experts nominated by the Member Countries was held to discuss revisions needed to the draft new OECD test guideline (TG), In Vitro Skin Corrosion Tests, in the light of comments received from Member Countries, and to agree on a revised draft version of the TG. For ease of updating, the initial draft guideline submitted to the OECD in December 1998 had been separated into two draft TGs, TG 430 (rat skin TER assay) and TG 431 (human skin model assays), in response to comments of the OECD National Coordinators in June The extended expert consultation meeting reached consensus on all of the technical issues discussed and on the revised wording of the two guidelines. The agreed drafts of TG 430 and TG 431 were circulated for agreement to the OECD Member Countries, and were finally approved in May 2002 at the 14th meeting of the OECD Working Group of National Coordinators of the Test Guidelines Programme. CORROSITEX CORROSITEX, one of the in vitro methods included in the ECVAM validation study, was subsequently evaluated by a peer-review panel convened by the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM [15]). The MT for the ECVAM validation study had concluded that CORROSI- TEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids (1). The database reviewed in the ICCVAM evaluation was considerably larger than that generated in the ECVAM prevalidation (10) and validation (1) studies, and the peer-review panel was able to be more specific in its conclusions, that: in limited testing situations the assay is valid for evaluating the corrosivity potential of acids, acid derivatives, and bases. The peer-review panel concluded that, in other testing situations, and for other chemical and product classes, the assay is appropriate as part of a tiered testing strategy (15). An ESAC statement on CORROSITEX, endorsing the ICCVAM findings, was agreed in December The ESAC concluded that the CORROSITEX assay is a scientifically validated test, but only for those acids, bases and their derivatives which meet the technical requirements of the assay (16). Skin irritation Dermal irritation is the production of reversible inflammatory changes in the skin (9). Information on the potential for chemicals to cause acute skin irritation is important when establishing procedures for the safe handling, packing and transport of chemicals, as well as for general safety-assessment purposes. The determination of acute skin irritation potential is therefore included in international regulatory requirements for the testing of chemicals (9). The standard approach for assessing skin irritation, based on the procedure of Draize et al. (17), involves applying a single dose of the test material (0.5ml or 0.5g) to a small area of the shaved skin of albino rabbits for up to 4 hours. The production of an irritant response (erythema and oedema formation) is determined by visual inspection of the skin and by scoring of the responses at 1, 24, 48 and 72 hours after patch removal. Observations may be continued for up to 14 days after dosing, to enable the reversibility of any effects to be fully evaluated (9). Testing for skin irritation in laboratory animals has the potential to cause them considerable discomfort or pain, and it is recognised that the response in the rabbit is not always predictive of that found in humans (7). For these reasons, several alternative (in vitro) methods for trying to identify skin irritants have been developed (7, 8; Table 1), the most promising of which appear to be reconstituted human skin models and animal (for example, pig, mouse) skin organ-culture methods (7). For chemical-induced acute skin irritation, the mode of application (i.e. topical) and exposure regimen (i.e. single dose applied for a short time) lend themselves to using non-animal methods that are able to mimic the route and time of exposure, and detect any subsequent acute tissue damage. As a follow-up to the successful validation of in vitro alternative tests for skin corrosion (1, 2), the ECVAM Skin Irritation Task Force recognised the urgent need to make progress in validating relevant in vitro tests for acute skin irritation (7). The Task Force initially reviewed a test protocol, prediction model and supporting data used with the EpiDerm human skin model, recommending to ECVAM that this test was put forward for prevalidation. In addition, since it was felt preferable to be able to include other in vitro tests in such a prevalidation study, the Task Force recommended that an open challenge was set, which involved laboratories submitting data on ten specified chemicals, and on 20% sodium lauryl sulphate as a reference standard, for

5 Alternative tests for skin irritation 65 review by the task force (7). Following review of test protocols, prediction models and data submitted by test developers, the Task Force recommended that ECVAM should support a prevalidation study on four tests: EpiDerm, EPISKIN, PREDISKIN (BIOPREDIC, Rennes, France) and the non-perfused pig ear method (6). Again, on the recommendation of the management team (MT) (following its review of the test protocol, prediction model and supporting data), an additional test, the mouse skin integrity function test (SIFT), was incorporated into the study in November 1999, following the completion of phase II with the four methods selected initially (6). Prevalidation A prevalidation study on in vitro tests for acute skin irritation (Table 1) was conducted during 1999 and 2000 (6). The study was principally funded by ECVAM, and was managed on behalf of ECVAM by Unilever s Safety and Environmental Assurance Centre (UK). The overall objective of validation in this area, of which the prevalidation study was an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to EU risk phrases ( R38 ; no classification) and the harmonised OECD criteria ( Irritant ; no label). The prevalidation study specifically addressed aspects of: protocol refinement (phase I), protocol transfer (phase II), and protocol performance (phase III), in accordance with the prevalidation scheme defined by ECVAM (12). The tests evaluated were: EpiDerm (phases I, II and III), EPISKIN (phases I, II and III), PRE- DISKIN (phases I and II, and additional protocol refinement), the non-perfused pig ear method (phases I and II, and additional protocol refinement), and the mouse SIFT (phases I and II; 6). A separate phase III study was conducted for the mouse SIFT (8), under the terms of an ECVAM contract with Syngenta s Central Toxicology Laboratory (UK). Modified, standardised test protocols and welldefined prediction models were available for each of the tests at the end of phase I. The results of phase I (intralaboratory reproducibility) were sufficiently promising for all of the tests to progress to phase II. Protocol transfer between the lead laboratory and a second laboratory was undertaken for all five tests during phase II, and additional refinements were made to the test protocols. For EpiDerm, EPISKIN and the SIFT, the intralaboratory and interlaboratory reproducibilities were acceptable; however, better standardisation of certain aspects of the test protocols was needed before commencing phase III. Neither PREDISKIN nor the pig ear test performed sufficiently well in phase II to progress to phase III. The PREDISKIN protocol was overly sensitive, resulting in the prediction of all the non-irritant (NI) chemicals as irritant (I). The variability in the pig ear test results was too great, indicating that the test would show limited predictive ability. In additional studies (a repeat of phase I), further modification of the PREDISKIN protocol and a change in the prediction model considerably improved the ability of the test to distinguish I from NI chemicals. However, attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. In phase III, an initial assessment of the reproducibility and predictive ability, in three independent laboratories per test, was undertaken for the EpiDerm and EPISKIN tests (subsequently, the SIFT was evaluated in a separate phase III study). A set of 20 coded chemicals (10 I, 10 NI) were tested with the final, refined, test protocols. The intralaboratory reproducibility was acceptable for both EpiDerm and EPISKIN. The interlaboratory reproducibility was considered to be acceptable for EPISKIN; however, for EpiDerm, an analysis of variance (ANOVA) indicated that there was a statistically significant laboratory effect on the overall variability, suggesting that the interlaboratory transferability of the test needs to be improved. The EpiDerm test had an overall accuracy of 58%, with an over-prediction rate of 37% and an under-prediction rate of 47%. The EPISKIN test had an overall accuracy of 58%, showing an under-prediction rate of 23% and an over-prediction rate of 60% (6). In the phase III study on the mouse SIFT, the overall accuracy of the test was 47%, with an over-prediction rate of 33% and an under-prediction rate of 73% (8). The MT concluded that none of the tests evaluated in the prevalidation study were yet ready for inclusion in a formal validation study on in vitro tests for acute skin irritation (6, 8). Follow-up to the ECVAM prevalidation study Although none of the five in vitro tests for skin irritation evaluated in the prevalidation studies met the performance criteria set by the MT for progression to a validation study, it was felt that appropriate modifications to some of the test protocols might enable them to enter the validation stage at some point in the future. Thus, various follow-up activities to the prevalidation study are currently in progress (8), primarily involving additional work on the EPISKIN (18), EpiDerm and SIFT test protocols and/or prediction models, with the objective of having test protocols suitable for inclusion in a formal validation study before the end of An extended ECVAM Skin Irritation Task Force meeting was held in May 2001, to agree and prioritise the activities needed before setting up a validation study (8). Improved protocols and prediction

6 66 J.H. Fentem & P.A. Botham models for the EpiDerm and EPISKIN (18) methods, the pig-ear test and the mouse SIFT were presented at the meeting. It was agreed that, in the short-term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a set of 20 new test chemicals. In addition, it was decided that the SIFT and pig ear test should be compared to see if common endpoints (transepidermal water loss [TEWL], methyl greenpyronine staining) could be identified. After the meeting, additional work was carried out on the two skin models, to evaluate whether they were comparable and whether it would be feasible to develop a single test protocol which could be applied to both models (8). The revised protocols and prediction models for the EPISKIN and EpiDerm tests for predicting acute skin irritation are close to meeting the acceptance criteria for progression to a validation study (8, 18), as defined by the MT for the prevalidation study (6). Re-analysis of the mouse SIFT phase III data by applying a new prediction model, suggested that the method is still promising, and should therefore be evaluated further with a new set of chemicals (8). Mouse or pig skin is also considered to be valuable for testing some hydrophilic compounds that penetrate the epidermis through hair follicles, which are lacking in reconstituted human skin models. It was suggested that laboratories using ex vivo pig or mouse skin with TEWL and/or electrical resistance (ER) endpoints should share their experience, with the aim of optimising and harmonising protocols, and also that methyl green-pyronine staining (a measure of cytotoxicity) should be investigated further for its applicability to reconstituted human skin models (8). New endpoints for predicting skin irritation in vitro, which could be easily measured with reconstituted human skin models such as EpiDerm and EPISKIN, were also discussed at the meeting (8). Since there are no obvious new candidate endpoints at present, it was concluded that further research to identify new markers for skin irritation, linked to a better mechanistic understanding, should be undertaken in the longer term (8). For this purpose, the application of genomics and/or proteomics technologies was considered to be the most promising approach (19). It was recommended that the research and development of the next generation of alternative methods for skin irritation involved further investigation of the utility of these tools for identifying more predictive markers and endpoints that could be readily measured in improved in vitro human skin models. Discussion The validation and regulatory acceptance of in vitro replacement alternative methods for skin corrosion testing, and the evaluation and prevalidation of in vitro tests for predicting acute skin irritation, have been reviewed in this paper. The role of ECVAM, in either managing and/or funding these studies, has been crucial to the considerable progress made. The validities of four in vitro tests for skin corrosion have been demonstrated, and these have been endorsed by the relevant European Commission services and their advisory committees, and by the Competent Authorities in the EU Member States. A new Test Method on Skin Corrosion (B.40) was incorporated into Annex V of Directive 67/548/EEC in mid-2000, making the use of in vitro replacement alternative methods for skin corrosion testing of chemicals mandatory in the EU. The OECD Member Countries have also now endorsed the rat skin TER and human skin model assays for skin corrosion, and these have been approved as test guidelines 430 and 431, respectively. Five in vitro methods for the prediction of acute skin irritation have undergone prevalidation in studies funded primarily by ECVAM. Of these, the EPISKIN, EpiDerm and mouse SIFT tests look the most promising, and it is hoped that these will be ready for inclusion in a formal validation study by the end of It is also widely recognised that new endpoints and test systems are required for predicting skin irritation in vitro, if in vivo studies are to be completely replaced. Further research is needed to identify new markers for skin irritation, linked to a better understanding of the mechanisms involved. In conclusion, it is apparent that ECVAM s activities in validating alternative tests for skin corrosion have resulted in the replacement of rabbit tests for this endpoint. Renewed efforts, collaboration and enthusiasm are now needed, if these achievements are to be repeated in the skin irritation area, where a validation study on the most promising in vitro tests available currently should be planned and undertaken during 2003 and Acknowledgement The authors would like to acknowledge the contributions to the studies reviewed here of all those involved in the ECVAM-funded skin corrosion validation and skin irritation prevalidation studies. References 1. Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhütter, H-G. & Liebsch, M. (1998). The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro 12, Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Uphill, P., Wilkins, S., Wiemann, C., Kaufmann, T., Remmele, M. & Holzhütter, H.G. (2000). The

7 Alternative tests for skin irritation 67 ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing. ATLA 28, Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G., de Silva, O., Holzhütter, H-G., Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J.M., Csato, M., Sladowski, D., Steiling, W. & Brantom, P. (1998). EU/COLIPA In vitro phototoxicity validation study, results of phase II (blind trial), part 1: the 3T3 NRU phototoxicity test. Toxicology in Vitro 12, Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., de Silva, O., Holzhütter, H-G., Gerberick, F., Liebsch, M., Lovell, W.W. & Pfannenbecker, U. (1998). A study on UV filter chemicals from Annex VII of European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test. ATLA 26, Barratt, M.D., Brantom, P.G., Fentem, J.H., Gerner, I., Walker, A.P. & Worth, A.P. (1998). The ECVAM international validation study on in vitro tests for skin corrosivity. 1. Selection and distribution of the test chemicals. Toxicology in Vitro 12, Fentem, J.H., Briggs, D., Chesné, C., Elliott, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Roguet, R., van de Sandt, J.J.M. & Botham, P.A. (2001). A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, Botham, P.A., Earl, L.K., Fentem, J.H., Roguet, R. & van de Sandt, J.J.M. (1998). Alternative methods for skin irritation testing: the current status. ECVAM Skin Irritation Task Force Report 1. ATLA 26, Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliott, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van de Sandt, J.J.M., Wiemann, C. & Worth, A.P. (2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ECVAM Skin Irritation Task Force Report 2. ATLA 30, OECD (1992) OECD Guidelines for the Testing of Chemicals, No. 404: Acute Dermal Irritation/ Corrosion, 6pp. Paris, France: OECD. 10. Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., Esdaile, D.J., Gardiner, J.R., Gordon, V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., Régnier, J-F., Steiling, W., Walker, A.P. & Balls, M. (1995). A prevalidation study on in vitro skin corrosivity testing. The report and recommendations of ECVAM workshop 6. ATLA 23, ECVAM (1998). Statement on the scientific validity of the rat skin transcutaneous electrical resistance (TER) test (an in vitro test for skin corrosivity). ATLA 26, Curren, R.D., Southee, J.A., Spielmann, H., Liebsch, M., Fentem, J.H. & Balls, M. (1995). The role of prevalidation in the development, validation and acceptance of alternative methods. ECVAM Prevalidation Task Force Report 1. ATLA 23, ECVAM (2000). Statement on the application of the EpiDerm human skin model for skin corrosivity testing. ATLA 28, EC (2000). Annex I to Commission Directive 2000/ 33/EC adapting to technical progress for the 27th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances. Official Journal of the European Communities L136, NIH (1999). CORROSITEX : an In Vitro Test Method for Assessing the Dermal Corrosivity Potential of Chemicals. NIH Publication No Research Triangle Park, NC, USA: NIEHS. 16. ECVAM (2001). Statement on the application of the CORROSITEX assay for skin corrosivity testing. ATLA 29, Draize, J.H., Woodard, G. & Calvery, H.O. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. Journal of Pharmacology and Experimental Therapeutics 82, Portes, P., Grandidier, M.H., Cohen, C. & Roguet, R. (2002). Refinement of the EPISKIN protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicology in Vitro, 16, Fletcher, S.T., Baker, V.A., Fentem, J.H., Basketter, D.A. & Kelsell, D.P. (2001). Gene expression analysis of EpiDerm following exposure to SLS using cdna microarrays. Toxicology in Vitro 15,