Skin Irritation Research – Part 1
Skin barrier disruption caused by organic solvents to human cadaver dermatomed skin was evaluated using an in vitro model system.
Resultant changes in transepidermal water loss (TEWL), as measured with an evaporimeter, were recorded after topical application of either acetone, chloroform:methanol 2:1, hexane, hexane:methanol 2:3, or the control, water, for exposure times of 1, 3, 6 and 12 min.
The resultant lipid/solvent mixture was removed and analyzed for its lipid content.
The ability of the different solvents to induce changes in the skin’s barrier function was assessed by comparing pre- to post-solvent exposure TEWL.
When compared to the controls, water and unexposed skin, chloroform:methanol 2:1 caused the greatest significant increase in TEWL, followed by hexane:methanol 2:3.
Acetone and hexane showed no difference in TEWL from the controls.
Besides solvent, exposure time was a significant independent variable for predicting change in TEWL, and the interaction of the two (exposure time and solvent type together) was the strongest predictor.
Lipid analysis of the extracts revealed that all the solvents removed comparable quantities of the surface lipids (triglycerides, wax esters, squalene, cholesterol esters).
Stratum corneum lipids–ceramides, free fatty acids, and cholesterol–extracted by chloroform:methanol 2:1 induced a significantly greater change in TEWL than hexane:methanol 2:3.
Additionally, no individual lipid class extracted by either chloroform:methanol 2:1 or hexane:methanol 2:3 proved to be a significant or accurate variable for predicting change in TEWL.
This suggests that the mechanism by which topical chloroform:methanol 2:1 and hexane:methanol 2:3 exposure induce a change in TEWL involves more than pure lipid extraction.
The principal objective of a skin irritation test is to determine the irritation potential of a substance so that a hazard assessment can be made and possible risk to humans evaluated.
The traditional test for evaluating skin irritation has been in vivo tests in the rabbit, often referred to as Draize tests.
hese have been employed for almost 50 years and are regarded as standard requirements for estimating the hazards associated with human skin exposure to test materials.
Since 1980, the validity and propriety of these tests have been increasingly questioned.
Much more attention has been given to the search for alternative test procedures in the hope that methods could be developed that would be both more humane and more predictive of human response.
RIFM wanted to investigate one of the new in vitro skin models for detecting skin irritation potential.
A key factor in effectively replacing animals with alternative methods is validation, which requires databases to correlate historical in vivo results with new in vitro results.
We had accumulated irritation data on approximately 100 fragrance materials using the procedure detailed in the EEC Directive and we felt that a study of representatives from this group of materials was a good place to start.
Skin2 was the model we investigated.
We chose MTT uptake, LDH release, and PGE2 release as endpoints to be measured.
Correlation of the results of these assays with those of the rabbit irritation test was not exact.
In vitro sensitisation assays have been proposed by various research groups and are actively being developed in our laboratories. The test described here is based on in vitro cultured dendritic-like cells (DCs) derived from human peripheral blood mononuclear cells.
These cells serve as replacement for Langerhans cells which are the most important antigen presenting cells in the skin.
After application of contact allergens to DCs, very rapid and selective increases of IL-1b mRNA expression followed by cell maturation have been reported (for example, Reutter et al. Toxicology in Vitro 11, 619-626, 1997 and Aiba et al. European Journal of Immunology 27, 3031-3038, 1997).
In the present work, the cell culture and the reverse transcriptase-polymerase chain reaction (RT-PCR) conditions have been optimized.
The expression of IL-1b mRNA has been measured 15 minutes after application of the test substance by a semi-quantitative RT-PCR procedure using some newly developed internal standards.
Maturation of the DCs observed as a time-dependent modulation of the CD86 positive cell population was then measured between 4 and 48 hours by flow cytometry.
Known irritants and sensitisers, as well as other potential sensitisers, were evaluated in this test format. Our results indicate that exposure to sensitisers but not to irritants induced reproducible increases in the CD86 positive cell population after 24 hours.
Specific and discriminating increases in IL-1b mRNA expression could also be observed in many, but not all, experiments.