Assessment of the sensitizing potency of preservatives with chance of skin contact by the loose-fit coculture-based sensitization assay (LCSA)
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Parabens, methylisothiazolinone (MI) and its derivative methylchloroisothiazolinone (MCI), are commonly used as preservatives in personal care products. They can cause hypersensitivity reactions of the human skin. We have tested a set of nine parabens, MI alone and in combination with MCI in the loose-fit coculture-based sensitization assay (LCSA). The coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DC-rc) in this assay emulates the in vivo situation of the human skin. Sensitization potency of the test substances was assessed by flow cytometric analysis of the DC-rc maturation marker CD86. Determination of the concentration required to cause a half-maximal increase in CD86-expression (EC50sens) allowed a quantitative evaluation. The cytotoxicity of test substances as indicator for irritative potency was measured by 7-AAD (7-amino-actinomycin D) staining. Parabens exhibited weak (methyl-, ethyl-, propyl- and isopropylparaben) or strong (butyl-, isobutyl-, pentyl- and benzylparaben) effects, whereas phenylparaben was found to be a moderate sensitizer. Sensitization potencies of parabens correlated with side chain length. Due to a pronounced cytotoxicity, we could not estimate an EC50sens value for MI, whereas MI/MCI was classified as sensitizer and also showed cytotoxic effects. Parabens showed no (methyl- and ethylparaben) or weak irritative potencies (propyl-, isopropyl-, butyl-, isobutyl-, phenyl- and benzylparaben), only pentylparaben was rated to be irritative. Overall, we were able to demonstrate and compare the sensitizing potencies of parabens in this in vitro test. Furthermore, we showed an irritative potency for most of the preservatives. The data further support the usefulness of the LCSA for comparison of the sensitizing potencies of xenobiotics.
KeywordsPreservatives Sensitizing potency In vitro sensitization assay LCSA Parabens
7-Amino-actinomycin D (CAS No. 7240-37-1)
Dendritic cell-related cells
Substance concentration causing a half-maximal increase in CD86-expression
Substance concentration causing 50 % cell death
Fluorescence-activated cell sorting
Fetal calf serum
Guinea pig maximization test
Keratinocyte growth medium 2
Loose-fit coculture-based sensitization assay
Local lymph node assay
Mean fluorescence intensity
Peripheral blood mononuclear cells
2,4,6-Trinitrobenzenesulfonic acid (CAS No. 2508-19-2)
Data presented in this publication are part of the master thesis of Anna Sonnenburg prepared during the master educational program in toxicology at Charité-Universitätsmedizin Berlin, supported by a grant from Berlin-Chemie AG, Berlin Germany. The LCSA is patented in Germany with the Application Number DE102007006736B4 (patent holder: Dagmar Briechle). Keratinocyte isolation from skin was performed with assistance of Mr. Sven Guhl at Clinic for Dermatology, Venerology and Allergology at Charité-Universitätsmedizin, Berlin. We are also grateful for a grant of the Sonnenfeld Stiftung, Berlin, providing financial support for the FACS Calibur.
Conflict of interest
The authors declare that they have no conflicts of interest.
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