Lube-Tech PUBLISHED BY LUBE: THE EUROPEAN LUBRICANTS INDUSTRY MAGAZINE
damage of the skin. However, to date no validated in vitro assay for testing and classification of skin irritation exists. However, in a pre- validation study by ECVAM for the testing of skin irritation and skin compatibility, human skin models turned out to be the most promising of several different in vitro methods (9). In addition to the investigations on alternative skin irritation models for classification purposes, several positive investigations on the testing of consumer products with low irritation potential on skin models also exist (10). In the experiments presented here we used an established in vivo study, the 24h Patch Test and an accepted in vitro test procedure on human skin models to test the skin compatibility of three cutting fluids. The aim was to find out whether these test systems are able to differentiate between the three test samples.
Experimental Materials
12 mm). Any irritant effects observed were recorded and documented 6, 24, 48 and 72 hours after removal of the plasters, divided into erythema, edema, squamation and fissuration parameters according to the scale of Frosch (11). The resulting score values are evaluated numbers reflecting the strength of the individual reactions. Individual score values were summed for all recorded time points and divided by the number of volunteers. The resulting total irritation score was calculated for each test substance, for erythema and a parameter combination of erythema + edema + squamation + fissuration (shown in tables). SDS 0.5% and demin. water were used reference substances for this test. For statistical comparison of the visual scores the “area under curve” (AUC) was calculated. A Many to One Comparison of product A and product C versus product B was performed. The Friedman test was used to clarify whether there were significant differences between the groups. The significance level was p<0.05.
Results and Discussion Three water soluble cutting fluids, A, B and C were analysed with regard to their skin compatibility in a 24h Patch Test and in skin models respectively. Differences in product formulas are listed in Table 1.
Cutting Fluids Table 1 lists the cutting fluids referred to in this paper. Product A, B and C are based on formulations of the MULTAN® series (Henkel technologies). General information on fields of application, components, pH, and use concentrations are listed.
Skin Models
SkinEthic human epidermis model (RHE) and SkinEthic human cornea model (HCE) were purchased from SkinEthic Laboratories (Nice, France). On arrival, cultures were transferred to fresh culture medium and incubated overnight at 37°C before application of the test samples.
Methods
Treatment of skin models A 30µl aliquot of test sample was applied directly onto the surface of the epithelial culture. Each product was tested in triplicates (minimum) against negative (water) and positive (0.5% or 1% SDS in water) controls.
Membrane Integrity Assay Media were sampled at the specified time intervals and tested for lactate dehydrogenase (LDH) activity. (Cytotoxicity Detection Kit LDH, Roche Diagnostic Corporation, Mannheim, Germany).
24 h Patch Test
The test was carried out on 20 healthy female and male volunteers in a mixed panel representing the average spectrum of the normal skin type. Volunteers were informed about the aim of the study, test procedure, test substances, and any possible health risks or discomforts before the start of the study. Written consent was signed by all volunteers before participating in the study. The test substances (5% dilution in demin. water) were applied side by side on the back of the volunteers over a period of 24 hours in a volume of 70ml under occlusive plasters (Finn Chamber on Scanpor,
2 LUBE MAGAZINE 79 JUNE 2007
In the in vitro tests, human epidermal skin models and human cornea models respectively, the products were tested with 5% and 10% product dilutions. Table 3 and Figures 1 and 2 show the LDH release in epidermis models after treatment with products A, B and C and with the positive control 1% SDS. An increase in LDH release indicates damage to the cell membrane leading to the leakage of enzymes from the cell. Not surprisingly, the 10% product dilution induced higher LDH-release compared to the 5% product dilution for all products tested. Clear differences between the three
The 5% product dilution used in the patch test resembles the recommended usage concentration for the products. At this concentration all products were well tolerated with irritation scores low above that of demin. water. Under these test conditions differences in skin compatibility between product A, B and C were not detectable. The Friedman test yielded no statistical significant differences in the score values of the three products. This finding corresponds with that of other investigators, who found patch tests on human volunteers insufficient to detect compatibility differences between well tolerated formulations (5).
In the 24h Patch Test the positive control (0,5% SDS) led to a total irritation score of 9.16. The negative control demin. water induced a total irritation score of 0.37. Products A, B and C induced irritation scores between 1.05 and 1.47. The irritation score for erythema was between 0.68 and 1.05 for products A, B and C. All irritation scores and related standard deviations are shown in Table 2.
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