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Development and validation of animal-free test methods to predict the skin sensitizing potential of chemicals

Bauch, Caroline D. (2013)
Development and validation of animal-free test methods to predict the skin sensitizing potential of chemicals.
Technische Universität Darmstadt
Ph.D. Thesis, Primary publication

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Item Type: Ph.D. Thesis
Type of entry: Primary publication
Title: Development and validation of animal-free test methods to predict the skin sensitizing potential of chemicals
Language: English
Referees: Schmitz, Prof. Dr. Katja ; Kimber, Prof. Dr. Ian
Date: 2013
Place of Publication: Darmstadt
Date of oral examination: 15 April 2013
Abstract:

Skin sensitization is the development of the allergic contact dermatitis caused by chemicals. Regulatory accepted methods to assess skin sensitizing potential of chemicals are animal based tests, but increasing interest in animal welfare presses the development of animal-free methods. The aim of this work was the development, establishment and validation of several alternative methods to animal testing to predict the skin sensitizing potential of chemicals. Therefore several methods reflecting different parts of the complex sensitization process have been used. Three steps of the skin sensitizing process were depicted: protein reactivity of chemicals, activation of keratinocytes and dendritic cell like cells have been investigated. Establishment and validation of the methods was performed with 54 test substances of known sensitizing potential, and the findings were compared to available human patch test data and murine local lymph node assay data. The experimental data were used to calculate the predictivity of each assay in order to compare the several assays and as well to evaluate possible combinations. By combination of different assays or more specifically the combination of the outcome of different assay into a testing strategy or testing battery predictivities were increased and allowed the prediction of sensitizing and non-sensitizing substances with high probability. The development of new methods to predict the protein reactive potential of sensitizing substances indicated that such compounds are able to react with proteins on the cell surface and that this reaction impacts the detection of such proteins by antibody staining. After treatment with sensitizing substances less protein was detectable and two hypotheses were proposed and investigated: It was shown that antibody binding was reduced or fully inhibited but also that the internalization of the altered protein was triggered. A correlation between the sensitizing potential and the altered protein level was confirmed although the underlying mechanism of how detectable protein level were reduced remained to be not fully understood. Further investigations are still required as the expression of the used protein in this study is up-regulated during the activation of dendritic cells. This phenomenon was observed for three out of five sensitizing substances. This work represents successful validations, inter- and intra-laboraty, of several animal-free test methods. The combinations of assays showed that predictivity compared to single assays can be increased and that for the number of tested substances these combinations are comparable to the local lymph node assay, the current gold standard to assess skin sensitizing potential. Although this work was no part of the formal validation of these assays, increased acceptance was gained, showing the reliability, reproducibility and high predictivities of these assays. The work on a new cell based protein reactivity assay showed a correlation between reduced level of detectable protein and the exposure of cells with sensitizers, although more investigation is required. The expression of the chosen protein unfortunately seemed to be up-regulated under the presence of skin sensitizer. Investigation of proteins their expression is independent of cell activation may provide more stable results.

Alternative Abstract:
Alternative AbstractLanguage

Die Entwicklung einer neuer Methoden zur Vorhersage des proteinreaktiven Potentials zeigte, das sensibilisierenden Substanzen mit Proteinen an der Zelloberfläche reagieren und Effekte auf die Detektion von Zelloberflächenmolekülen mittels Antikörperfärbung haben können. Nach Behandlung der Zellen mit sensibilisierenden Substanzen konnte weniger Protein an der Zelloberfläche festgestellt werden. Hierzu wurden zwei unterschiedliche Hypothesen aufgestellt und experimentell überprüft. Es wurde gezeigt, dass die Reaktion von Chemikalien mit Proteinen an der Zelloberfläche die Bindung von Antikörpern reduziert oder vollständig inhibiert, aber auch die Internalisierung des veränderten Proteins auslöst. Ein Zusammenhang zwischen der sensibilisierenden Eigenschaft von Chemikalien und der reduzierten nachweisbaren Proteinmenge wurde gezeigt, obwohl der Mechanismus welcher der Reduktion des nachweisbaren Proteins zu Grunde liegt nicht vollständig geklärt werden konnte. Nicht sensibilisierende Substanzen hatten keinen Effekt auf den nachweisbaren Proteingehalt. Weitere Untersuchungen sind nötig, da die Expression des untersuchten Proteins mit Aktivierung dendritischer Zellen hochreguliert werden kann. Diese Phänomen wurde bei drei von fünf sensibilisierenden Substanzen beobachtet. Diese Arbeit zeigte die erfolgreiche Validierungen verschiedener versuchstierfreier Methoden, welche zum einen intern aber auch teilweise mit externen Laboren als Ringstudien durch geführt wurde. Es hat sich gezeigt, dass die Kombination dieser Methoden die Aussagekraft im Vergleich zu den einzelnen Methoden erhöht hat und, für die Anzahl der getesteten Substanzen, vergleichbare Werte zum Standardtest, dem Lokalen Lymphknoten Test, erzielt wurden. Obwohl diese Ringversuche nicht Teil der offiziellen Validierung der einzelnen Tests waren, wurde die Anerkennung durch die gezeigte Zuverlässigkeit, Reproduzierbarkeit und hohe Prädiktivitäten erhöht. Die Entwicklung eines neuen zellbasierten Tests zur Untersuchung der Proteinreaktivität zeigte, dass es einen Zusammenhang zwischen der erniedrigten, messbaren Menge des Proteins und der Behandlung der Zellen mit Sensibilisierer gibt, dennoch sind weitere Experimente nötig. Die Expression des gewählten Proteins wurde teilweise durch die Behandlung mit Sensibilisierer hoch reguliert. Proteine, deren Expression unabhängig von aktivierten Zellen ist, ergeben möglicherweise stabilere Ergebnisse.

German
Alternative keywords:
Alternative keywordsLanguage
Skin sensitization, 3R concept, Cosmetic directive, alternavite methodsEnglish
URN: urn:nbn:de:tuda-tuprints-35136
Classification DDC: 500 Science and mathematics > 540 Chemistry
500 Science and mathematics > 570 Life sciences, biology
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Clemens-Schöpf-Institut > Fachgebiet Biochemie
Date Deposited: 29 Jul 2013 07:25
Last Modified: 09 Jul 2020 00:29
URI: https://tuprints.ulb.tu-darmstadt.de/id/eprint/3513
PPN: 386800537
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