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Archives of Toxicology

, Volume 89, Issue 12, pp 2339–2344 | Cite as

Assessment of the sensitizing potency of preservatives with chance of skin contact by the loose-fit coculture-based sensitization assay (LCSA)

  • Anna SonnenburgEmail author
  • Maximilian Schreiner
  • Ralf Stahlmann
Immunotoxicology

Abstract

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.

Keywords

Preservatives Sensitizing potency In vitro sensitization assay LCSA Parabens 

Abbreviations

7-AAD

7-Amino-actinomycin D (CAS No. 7240-37-1)

DC

Dendritic cells

DC-rc

Dendritic cell-related cells

DMSO

Dimethylsulfoxide

EC50sens

Substance concentration causing a half-maximal increase in CD86-expression

EC50vit

Substance concentration causing 50 % cell death

FACS

Fluorescence-activated cell sorting

FCS

Fetal calf serum

GMPT

Guinea pig maximization test

KC

Keratinocytes

KGM-2

Keratinocyte growth medium 2

LCSA

Loose-fit coculture-based sensitization assay

LLNA

Local lymph node assay

MFI

Mean fluorescence intensity

MCI

Methylchloroisothiazolinone

MI

Methylisothiazolinone

PBMC

Peripheral blood mononuclear cells

PE

Phycoerythrin

TNBS

2,4,6-Trinitrobenzenesulfonic acid (CAS No. 2508-19-2)

Notes

Acknowledgments

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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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Anna Sonnenburg
    • 1
    Email author
  • Maximilian Schreiner
    • 2
  • Ralf Stahlmann
    • 1
  1. 1.Institute of Clinical Pharmacology and ToxicologyCharité - Universitätsmedizin BerlinBerlinGermany
  2. 2.Department of Internal MedicineBundeswehr HospitalBerlinGermany

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