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


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.


Preservatives Sensitizing potency In vitro sensitization assay LCSA Parabens 



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


Dendritic cells


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.


  1. Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay RB, Landsiedel R (2012) Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol 63:489–504CrossRefPubMedGoogle Scholar
  2. Caon T, Costa AC, de Oliveira MA, Micke GA, Simoes CM (2010) Evaluation of the transdermal permeation of different paraben combinations through a pig ear skin model. Int J Pharm 391:1–6CrossRefPubMedGoogle Scholar
  3. Carradori S, Peluso AM, Faccioli M (1990) Systemic contact dermatitis due to parabens. Contact Dermatitis 22:238–239CrossRefPubMedGoogle Scholar
  4. Cashman AL, Warshaw EM (2005) Parabens: a review of epidemiology, structure, allergenicity, and hormonal properties. Dermatitis 16:57–66PubMedGoogle Scholar
  5. European Union (2014) Official J European Union. L 107/5.
  6. Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY, Basketter DA (2005) Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis 16:157–202PubMedGoogle Scholar
  7. Karpuzoglu E, Holladay SD, Gogal RM Jr (2013) Parabens: potential impact of low-affinity estrogen receptor binding chemicals on human health. J Toxicol Environ Health B Crit Rev 16:321–335CrossRefPubMedGoogle Scholar
  8. Lundov MD, Moesby L, Zachariae C, Johansen JD (2009) Contamination versus preservation of cosmetics: a review on legislation, usage, infections, and contact allergy. Contact Dermatitis 60:70–78CrossRefPubMedGoogle Scholar
  9. Lundov MD, Krongaard T, Menne TL, Johansen JD (2011) MI contact allergy: a review. Br J Dermatol 165:1178–1182CrossRefPubMedGoogle Scholar
  10. Maio P, Carvalho R, Amaro C, Santos R, Cardoso J (2012) Contact allergy to methylchoroisothiazolinone/methylisothiazolinone (MCI/MI): findings from a Contact Dermatitis Unit. Cutan Ocul Toxicol 31:151–153CrossRefPubMedGoogle Scholar
  11. McFadden JP, Basketter DA (2000) Contact allergy, irritancy and ‘danger’. Contact Dermatitis 42:123–127CrossRefPubMedGoogle Scholar
  12. N.N. (2008) Final amended report on the safety assessment of methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, isobutylparaben, and benzylparaben as used in cosmetic products. Int J Toxicol 27 Suppl 4:1–82Google Scholar
  13. Nohynek GJ, Borgert CJ, Dietrich D, Rozman KK (2013) Endocrine disruption: fact or urban legend? Toxicol Lett 223:295–305CrossRefPubMedGoogle Scholar
  14. Schnuch A, Lessmann H, Geier J, Uter W (2011a) Contact allergy to preservatives. analysis of IVDK data 1996–2009. Br J Dermatol 164:1316–1325CrossRefPubMedGoogle Scholar
  15. Schnuch A, Mildau G, Kratz EM, Uter W (2011b) Risk of sensitization to preservatives estimated on the basis of patch test data and exposure, according to a sample of 3541 leave-on products. Contact Dermatitis 65:167–174CrossRefPubMedGoogle Scholar
  16. Schreiner M, Peiser M, Briechle D, Stahlmann R, Zuberbier T, Wanner R (2007) A loose-fit coculture of activated keratinocytes and dendritic cell-related cells for prediction of sensitizing potential. Allergy 62:1419–1428CrossRefPubMedGoogle Scholar
  17. Schreiner M, Peiser M, Briechle D, Stahlmann R, Zuberbier T, Wanner R (2008) A new dendritic cell type suitable as sentinel of contact allergens. Toxicology 249:146–152CrossRefPubMedGoogle Scholar
  18. Soni MG, Burdock GA, Taylor SL, Greenberg NA (2001) Safety assessment of propyl paraben: a review of the published literature. Food Chem Toxicol 39:513–532CrossRefPubMedGoogle Scholar
  19. Sonnenburg A, Ahuja V, Schreiner M, Platzek T, Stahlmann R (2012) Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by LCSA. Arch Toxicol 86:733–740CrossRefPubMedGoogle Scholar
  20. Thyssen JP, Engkilde K, Lundov MD, Carlsen BC, Menne T, Johansen JD (2010) Temporal trends of preservative allergy in Denmark (1985–2008). Contact Dermatitis 62:102–108CrossRefPubMedGoogle Scholar
  21. Toncic RJ, Lipozencic J, Martinac I, Greguric S (2011) Immunology of allergic contact dermatitis. Acta Dermatovenerol Croat 19:51–68PubMedGoogle Scholar
  22. Uter W, Yazar K, Kratz EM, Mildau G, Liden C (2014) Coupled exposure to ingredients of cosmetic products: II. Preservatives. Contact Dermatitis 70:218–226Google Scholar
  23. Wanner R, Schreiner M (2008) An in vitro assay to screen for the sensitizing potential of xenobiotics. ALTEX 25:115–120PubMedGoogle Scholar
  24. Wanner R, Sonnenburg A, Quatchadze M, Schreiner M, Peiser M, Zuberbier T, Stahlmann R (2010) Classification of sensitizing and irritative potential in a combined in vitro assay. Toxicol Appl Pharmacol 245:211–218CrossRefPubMedGoogle Scholar

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

Personalised recommendations