Multi-laboratory evaluation of SkinEthic HCE test method for testing serious eye damage/eye irritation using solid chemicals and overall performance of the test method with regard to solid and liquid chemicals testing.

A prospective multicentre study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITS protocol for solids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 35 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 95% (57/60) and 96.8% (92/95) for the extended data set. Furthermore, the overall concordance between the laboratories was 96.7% (58/60). The accuracy of the SkinEthic™ HCE EITS for the extended dataset, based on bootstrap resampling, was 81.0% (95% CI: 78.9% to 83.2%) with a sensitivity of 90.5% (95% CI: 88.1% to 92.9%) and specificity of 73.6% (95% CI: 71.7% to 75.5%). Overall, 200 chemicals were tested (105 liquids (EITL protocol) and 95 solids (EITS protocol)) resulting in a sensitivity of 95.2%, specificity of 72.1% and accuracy of 83.7%, thereby meeting all acceptance criteria for predictive capacity.

Multi-laboratory validation of SkinEthic HCE test method for testing serious eye damage/eye irritation using liquid chemicals.

A prospective multicentric study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITL protocol for liquids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 45 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 88.3% (53/60) and 92.4% (97/105) for the extended data set. Furthermore, the overall concordance between the laboratories was 93.3% (56/60). The accuracy of the SkinEthic™ HCE EITL for the extended dataset, based on bootstrap resampling, was 84.4% (95% CI: 81.9% to 87.6%) with a sensitivity of 99.0% (95% CI: 96.4% to 100%) and specificity of 68.5% (95% CI: 64.0% to 74.0%), thereby meeting all acceptance criteria for predictive capacity. This efficient transferable and reproducible assay is a promising tool to be integrated within a battery of assays to perform an eye irritation risk assessment.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Percutaneous penetration of 2-phenoxyethanol through rat and human skin.

2-Phenoxyethanol applied in methanol was absorbed (64 +/- 4.4% at 24 hr) through unoccluded rat skin in vitro in the static diffusion cell with ethanol/water as receptor fluid. By comparison (43 +/- 3.7% in 24 hr) was absorbed in the flow-through diffusion system with tissue culture medium as receptor fluid. 2-Phenoxyethanol applied in methanol was absorbed (59.3 +/- 7.0% at 6 hr) through unoccluded human skin in vitro in the flow-through diffusion cell with tissue culture medium. With both unoccluded cells, 2-phenoxyethanol was lost by evaporation but occlusion of the static cell reduced evaporation and increased total absorption to 98.8 +/- 7.0%. Skin, post mitochondrial fraction, metabolized phenoxyethanol to phenoxyacetic acid at 5% of the rate for liver. Metabolism was inhibited by 1 mM pyrazole, suggesting involvement of alcohol dehydrogenase. However, first-pass metabolism of phenoxyethanol to phenoxyacetic acid was not detected during percutaneous penetration through viable rat skin in the flow-through system. First-pass metabolism in the skin does not therefore have an influence on systemic availability of dermally absorbed phenoxyethanol. These measures of phenoxyethanol absorption through rat and human skin in vitro agree well with those obtained previously in vivo.

Prediction of the percutaneous penetration and metabolism of dodecyl decaethoxylate in rats using in vitro models.

Percutaneous absorption of a lipophilic surfactant, dodecyl decaethoxylate, can be predicted using in vitro models. In vivo, dermal penetration of dodecyl decaethoxylate was found to be 22.9% in 48 h. All of the absorbed dodecyl decaethoxylate in the rat was metabolised and excreted in expired air as carbon dioxide, or in the urine and faeces. Using rat skin mounted in the unoccluded flow-through diffusion cell with MEM as receptor fluid, in vivo absorption was predicted by the percentage of the applied dose recovered in the stratum corneum, epidermis, dermis and receptor fluid at 24 h (25%). Conversely, the penetration of dodecyl decaethoxylate was over-predicted in the unoccluded static diffusion cell using aqueous ethanol (50% v/v) as the receptor fluid where 49.4% recovered in the receptor fluid at 24 h. In vitro models may be used to predict percutaneous absorption and reduce animal use, provided a suitable receptor fluid is used in which the penetrant is soluble. Dermal metabolism of dodecyl decaethoxylate was low and not considered to influence dermal absorption.