In vitro assessment of skin sensitisation potential

Genomic biomarker analysis for sensitisation prediction

This is one of four test methods (DPRA, KeratinoSensTM and h-CLAT) for the assessment of skin sensitisation potential.

Assessment of skin sensitisation potential with the GARDTM

  • The third molecular key event of skin sensitisation addresses the activation of dendritic cells (DC), which is typically accompanied by expression of specific cell surface markers, chemokines and cytokines.
  • The GARDTM assay can be performed to predict the ability of chemicals to induce skin sensitisation based on the analysis of relative expression levels of a biomarker signature of 196 genes. These biomarkers represent the various activated mechanisms in dendritic cells (DCs) in response to chemicals. The test method uses SenzaCell, a human myeloid leukemia cell line. This cell line works as an in vitro model of human dendritic cells and chemical stimulation of the cells can be assessed.
  • With this test method transcriptional quantification of the genomic predictors, termed collectively the GARDTM Prediction Signature (GPS), using a Nanostring nCounter technology can be determined. Based on a derived decision value (DV) from a Support Vector Machine (SVM) model chemicals can be predicted to be sensitisers or non-sensitisers. The algorithm of this machine uses known data sets from already done cell stimulations with known chemicals. Using this data set for each new chemical a decision value is created and the mean value is used for classification. A sensitising chemical can be further classified into category 1A or 1B.
  • All experimental steps until the isolation of the RNA are performed at the Eurofins Human Safety Testing laboratory. The qualification of the RNA and the Nanostring endpoint measurement are performed in cooperation with SenzaGen AB in Sweden.
  • In combination with other complementary information within an Integrated Approach to Testing and Assessment (IATA) or as a stand-alone method, the GARDTM Assay can be used as a reliable in vitro method to assess skin sensitising potential of chemicals.

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sensitization-card

"The GARDTM method mimics the immune system by using human dendritic cells. It predicts the ability of chemical compounds to induce skin sensitisation by measuring changes in the genomic profile of the cells after chemical treatment."

Principles of the GARD™ assay

Protocol

  

Cell line
  • SenzaCells provided by SenzaGen AB
  • Human myeloid leukemia cell line

Analysis

 Cell viability and assessment of genomic biomarkers
Test chemical concentrations  
  • Input finder: 9 concentrations with a minimum of 1 mM
  • Main stimulation: 1 concentration leading to a cell viability of 90 ± 5%
Incubation time

 24 h ± 0.5 h at 37 °C and 5% CO2
Quality controls  
  • Medium control
  • Negative control: 0.1% DMSO
  • Positive control: 75 µM p-phenylene diamine (PPD)
Solvents of test chemical  
  • Dimethylsulfoxid (DMSO)
  • Water
Application

 At least three independent performed experiments
Data delivery  
  • Cell viability with FACS analysis
  • Nanostring data analysis using GARD Data Analysis Application (SenzaGen AB)
Classification  
  • Mean DV of biological replicate samples ≥0 = sensitiser
  • Mean DV of biological replicate samples <0 = non-sensitiser

References

  1. The OECD (2012), The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168
  2. UN (2015), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Sixth revised edition, UN New York and Geneva