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.