Skin2--an in vitro human skin model: the correlation between in vivo and in vitro testing of surfactants.

The availability of an in vitro test system to replace animal testing of potential irritants is becoming more and more urgent especially in Europe as a consequence of the European Community Cosmetics Directive. To evaluate the ability of Advanced Tissue Sciences' (ATS) ZK1301 skin model to predict the skin irritation potential of surfactants, we performed a pilot validation study utilizing four different laboratories. The in vitro protocol was designed as a quantitative pre-screen for the clinical patch studies. Sixteen substances, representing various surfactant categories and ranges of irritation potential, were tested. The 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantitate viability in vitro. We documented the viability of tissues exposed to unknown substances for specific periods. The in vitro results were calculated as percent distilled water controls (DWC). The time required to reduce the viability of each tissue to 50% of the distilled water controls (T50) was compared to mean erythema and edema scores from the clinical studies by Pearson's correlation. The individual laboratories demonstrated coefficients of 0.72. The results indicated that the 30 min percent untreated control values best predicted the 24 h clinical patch scores. No statistically significant interlab variability was found. Only one false negative was seen when non/mild and moderate/severe irritant categories were assigned according to the in vitro scores. These results demonstrate that the skin2 in vitro test system may serve as a good screening method prior to clinical patch studies.

Comparison of low-volume, Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations.

The first phase in a series of investigations of the relationship between low-volume eye test (LVET) data, Draize eye irritation test data, and comparable data from 25 in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro assay data generated previously as part of the Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program. LVET data were generated de novo using the same 10 representative hydroalcoholic personal-care formulations. The linear correlation between maximum average score (MAS) as determined by the Draize test and the LVET (LVET-MAS) was 0.93. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. As in the CTFA program, regression modelling is the primary means of enabling such a comparison. The objective is to predict LVET-MAS for a given test material (and to place upper and lower prediction interval bounds in the range in which the LVET-MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Four assays [EYTEX MPA (membrane partition assay), HET-CAM (hen's egg test-chorioallantoic membrane HET-CAM) I, neutral red release and HET-CAM II] were shown to have the greatest agreement with the LVET. These assays were also among those with low discordance rates relative to the Draize test. Prediction of LVET-MAS values from experimentally determined in vitro scores was more accurate for hydroalcoholic formulations with lower rather than higher irritancy potential.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II) oil/water emulsions.

The Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program is an evaluation of the relationship between Draize ocular safety test data and comparable data from a selection of in vitro tests. In Phase II, 18 representative oil/water-based personal-care formulations were subjected to the Draize primary eye safety test and 30 in vitro assay protocols (14 different types of in vitro endpoints were evaluated; the remainder were protocol variations). Correlation of in vitro with in vivo data was evaluated using analysis of sensitivity/specificity and statistical analysis of the relationship between maximum average Draize score (MAS) and in vitro endpoint. Regression modelling is the primary approach adopted in the CTFA Program for evaluating in vitro assay performance. The objective of regression analysis is to predict MAS for a given test material (and to place upper and lower prediction interval bounds on the range in which the MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves: the narrower the prediction interval, the more predictive of the Draize score is the in vitro test result. 16 assays were shown to have the greatest agreement with the Draize procedure and were therefore selected for regression analysis. Based on the magnitude of the 95% prediction bounds of each of the 16 selected assays over the range of test data, it may be inferred that prediction of MAS values from experimentally determined in vitro scores is more accurate for oil/water-based formulations with lower rather than higher irritancy potential. The assays selected for modelling in Phase II generally exhibited weaker relationships with MAS than those selected in Phase I (evaluated using hydroalcoholic formulations), even though several assays were common to both Phases.

Percutaneous penetration of N-nitrosodiethanolamine through human skin (in vitro): comparison of finite and infinite dose applications from cosmetic vehicles.

N-Nitroso compounds (nitrosamines) have been detected at the parts per billion level in a wide variety of matrices including industrial chemicals, pharmaceuticals, and food. Although N-nitrosodiethanolamine (NDELA) may be detected as an impurity in some cosmetic products, studies on NDELA absorption through human skin have been limited. A study to determine the extent of NDELA absorption following topical application was therefore undertaken to assist in the proper assessment of risk following unintended exposure. NDELA absorption was measured in vitro through human cadaver skin using isopropyl myristate (IPM) and generic prototype personal-care formulations (sunscreen and shampoo) spiked with [14C]NDELA. When applied as a finite dose at a concentration of 0.06% or lower, NDELA absorption was found to be a linear function of concentration. Total absorption at 48 hr ranged from approximately 35 to 65% of the dose and was formulation dependent (IPM > shampoo > or = sunscreen). Absorption occurred relatively rapidly from all formulations and peak rates of absorption were seen within the first 5 hr from the IPM and shampoo formulations. When applied as an infinite dose, total NDELA absorption followed a different rank order (shampoo > or = IPM > sunscreen) and evidence of barrier damage was seen with the shampoo formulation.

Prediction of the eye irritation potential of shampoos using the in vitro SIRC cell toxicity test.

Shampoos predicted to vary over a wide range in their potential to cause eye irritancy were tested in SIRC rabbit corneal cells for cytotoxicity. Six shampoos at at least ten different concentrations were added to culture dishes containing the SIRC cells and were administered at a dose of 0.01 ml directly to the corneal surface of the albino rabbit eye. When the shampoos were ranked both according to their relative toxicities in vitro and according to their relative irritancies in the modified Draize test the resulting rank orders were in perfect agreement. The results demonstrate that the SIRC cell line is a suitable model system for screening shampoos of differing surfactant compositions without the need to use live animals. Further, results obtained from the SIRC cell assay should aid in the selection of final product formulations for in vivo testing as part of the pre-market clearance process.

Deposition of 3,4,4'-trichlorocarbanilide on human skin.

For rinse-off products such as soaps, quantitating the deposition of ingredients left behind on the skin following rinsing may be important particularly if the ingredient is biologically active, e.g. germicides, perfumes. The residue comprises the major portion of material which is available for percutaneous penetration. A method is described for quantitating the deposition of the soap germicide, 3,4,4'-trichlorocarbanilide (TCC) as the result of direct application of bar soap on human skin. A soap pellet containing 1.5% [14C]TCC was gently rubbed on a small area of the volar forearm for 30 s. 90 s after soap application the site was thoroughly rinsed with water. After rinsing, 1.4% of the applied [14C]TCC, or 0.33 micrograms/cm2, remained on the treated site as determined by cellophane tape stripping followed by scintillation counting. The amount of TCC deposited from a simulated wash with a bar soap onto human skin is compared to the amount deposited onto the skin of other species.

The hairless mouse as a model for quantitating skin deposition of 3,4,4'-trichlorocarbanilide in bar soap.

A method is described for quantitating the deposition of the germicide 3,4,4'-trichlorocarbanilide (TCC) via direct application of bar soap to the skin. The soap contained 1.5% [14C]TCC. Quantitating the skin deposition of biologically active materials is important in the safety evaluation of these ingredients as well as the finished products. In the case of rinse-off products such as soaps, the residue remaining after rinsing constitutes the major portion of material available for penetration. The hairless mouse and the clipped albino Sprague-Dawley rat were evaluated as models for human skin deposition. Little TCC remained on the skin of either species following the wash and rinse procedure. The amount deposited on rat skin was 1.5% of the applied dose or 0.87 micrograms TCC/cm2 while the amount deposited on hairless mouse skin was 1.1% or 0.18 micrograms TCC/cm2. The greater deposition of TCC onto rat skin was likely to be due to the presence of a greater amount of hair. Results obtained using the hairless mouse were consistent and reproducible. The hairless mouse does not require shaving and is easy to handle. Since, like man, it has little hair, it appears to be an excellent model for use in predicting the deposition of TCC on human skin.

Evaluation of an in vitro cell toxicity test using rabbit corneal cells to predict the eye irritation potential of surfactants.

Surfactants reported to be severely, moderately or non-irritating to the eyes of albino rabbits were tested for cell toxicity in vitro using an established line of rabbit corneal cells. Test substances at different concentrations were added to culture dishes containing the rabbit corneal cells or were administered at a dose of 0.01 ml directly to the corneal surface of albino rabbits. A significant rank correlation (r = 0.90, P less than 0.01) was seen between the relative toxicity produced in vitro and the relative eye irritation produced in the modified Draize test. The in vitro method offers a means for screening potentially irritating chemicals without using live animals and may be useful in the selection of product formulations for in vivo testing.