No abstract

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, inter-leukin-1α (IL-1α), 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-1α 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-1α 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-1α 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.

No abstract

The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emissions, to affect cell viability, thiol status and intracellular Ca2+ levels and to cause DNA damage and mutations has been studied using cultured human cells. Within a concentration range of 3-100 mM, a 1 h exposure to acetaldehyde decreases colony survival and inhibits uptake of the vital dye neutral red in bronchial epithelial cells. Acetaldehyde also causes both DNA interstrand cross-links and DNA protein cross-links whereas no DNA single strand breaks are detected. The cellular content of glutathione is also decreased by acetaldehyde, albeit, without concomitant changes in the glutathione redox status or in the content of protein thiols. Transient or sustained increases in cytosolic Ca2+ occur within minutes following exposure of cells to acetaldehyde. Moreover, acetaldehyde significantly decreases the activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Finally, a 5 h exposure to acetaldehyde causes significant levels of 6-thioguanine resistance mutations in an established mutagenesis model involving skin fibroblasts. The results indicate that mM concentrations of acetaldehyde cause a wide range of cytopathic effects associated with multistep carcinogenesis. The fact that acetaldehyde, in relation to its cytotoxicity, causes comparatively higher genotoxicity and inhibits DNA repair more readily than other major aldehydes in tobacco smoke and automotive emissions is discussed.

Experience has shown that the outcome of large and expensive validation studies on alternative methods can be compromised if their managers do not insist that optimised test protocols and proof of their performance are submitted before the start of the formal validation study. One way for the sponsors of validation studies to confirm both the likely relevance of a method for its stated purpose and its readiness for validation would be to require a prevalidation study before formal validation was contemplated. This process would involve the developers (or other proponents of the method) and selected independent laboratories in protocol refinement (Phase I) and protocol transfer (Phase II). The optimised protocol would then be assessed in a protocol performance phase (Phase III), which would involve the testing of a relevant set of coded test materials and an evaluation of a proposed prediction model. In certain circumstances, a successful outcome of Phase III might be sufficient for promotion of the regulatory acceptance of the method. Normally, however, the method would proceed to a formal validation study. The European Centre for the Validation of Alternative Methods, a recognised validation authority, now proposes to introduce this prevalidation scheme into its validation strategy.

No abstract

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm™ method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN™ and PREDISKIN™, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999–2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. 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 new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig-ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

Formaldehyde, a common environmental pollutant, inhibits repair of O6-methylguanine and potentiates the mutagenicity of an alkylating agent, N-methyl-N-nitrosourea, in normal human fibroblasts. Because formaldehyde alone also causes mutations in human cells, the compound may cause genotoxicity by a dual mechanism of directly damaging DNA and inhibiting repair of mutagenic and carcinogenic DNA lesions caused by other chemical and physical carcinogens.