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Characterization of the sensitizing
potential of chemicals by in vitro analysis of dendritic cell activation and skin
penetration Dr.
Carsten Goebel, Wella AG, Abteilung Produktsicherheit und Toxikologie, Darmstadt
The development of in vitro models to identify sensitizing chemicals received
public interest since animal testing should be avoided whenever possible. We introduced
a new approach, analyzing two essential properties of sensitizing chemicals: skin
penetration and dendritic cell (DC) activation (J Invest Dermatol 122:1154 –1164,
2004). Immature DCs derived from human peripheral blood monocytes were pooled
from different donors to reduce interindividual variation. DC activation was evaluated
by flow cytometric analysis of CD86 highly positive cells and quantitative measurement
of interleukin-1ß (IL-1ß) and Aquaporin P3 gene expression. The sensitizer
2,4,6-trinitrobenzenesulfonic acid (TNBS) induced a dose dependent response for
all three parameters, whereas the irritant sodium lauryl sulfate (SDS) did not.
When two related aromatic amines, p-toluylenediamine (PTD) and hydroxyethyl-p-phenylenediamine
(HE-PPD), were tested, both induced a substantial DC activation indicating their
potential sensitizing properties. However, the in vitro findings contrasted with
the in vivo sensitizing potential: in murine local lymph node assays (LLNA) PTD,
but not HE-PTD,was identified as sensitizing using acetone/aqua/olive oil (AAOO)
as vehicle. Skin penetration measurement revealed that the bioavailability of
PTD from AAOO was significantly higher than that of HE-PPD. An enhanced skin penetration
of HE-PPD was found when dimethysulfoxide (DMSO) was used as vehicle. On retesting
HE-PPD in the LLNA using DMSO as vehicle, HE-PPD induced a specific response reflecting
its activating properties on DCs in vitro. We conclude that in vitro analysis
of DC activation capability allows prediction of the skin sensitizing potential
provided that skin penetration data demonstrate a sufficient bioavailability of
the test compound.
Background
Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity reaction
induced by small reactive chemicals (haptens). In pharmaceutical and cosmetic
industry it is mandatory to identify chemicals that are potential ACD inducers
before they become part of a new product. Currently, the sensitizing potential
of chemicals is usually identified on the basis of animal studies, such as the
LLNA. There is, however, an increasing public concern regarding the use of animal
testing for the screening of new chemicals. The development of in vitro models
for predicting the sensitizing potential of new chemicals is therefore receiving
widespread interest. In vitro sensitization tests are furthermore needed to identify
the relevant aspects of the complex interactions of a chemical with the different
compartments of the immune system. To date, no a reliable prediction
of the sensitizing potential based on DC activation in vitro was achieved because
the in vitro results did not reflect the actual ability to induce contact hypersensitivity
in vivo. We assume that the lack of the skin barrier and the difficulties
in obtaining relevant DC activation data are responsible for this discrepancy.
Therefore, we propose to integrate in vitro skin penetration analysis using pig
skin, the most suitable model of human skin, and to use pooled immature DCs obtained
from different donors to reduce inter-individual variability which limit the interpretation
of DC activation data. The suitability of this approach was tested with two aromatic
amines, a class of chemicals comprising several known sensitizers. The in vitro
findings for the two compounds were compared with their potential to induce sensitization
in vivo as measured by the LLNA in mice. nach oben |