A Comparative Evaluation of In Vitro Skin Sensitisation Tests: The Human Cell-line Activation Test (h-CLAT) versus the Local Lymph Node Assay (LLNA)

Abstract

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential.

Artificial neural network analysis of data from multiple in vitro assays for prediction of skin sensitization potency of chemicals

“…Although various in vitro skin sensitization assays, including h-CLAT and a peptide-binding assay (direct peptide reactivity assay; DPRA), have been developed (Ashikaga et al, 2010;Gerberick et al, 2005), little work has been done to build prediction models for the skin sensitization potential of chemicals in vivo. However, some reports have mentioned relationships among EC3 values from LLNA and individual in vitro assays (Gerberick et al, 2004a,b;Natsch and Emter, 2008).…”

“…We selected 64 test chemicals for which LLNA and h-CLAT data are available in the literature (Ashikaga et al, 2010;Basketter, 2010Basketter, , 2007Basketter and Kimber, 2011;Basketter and Scholes, 1992;Hoya et al, 2009;Ryan et al, 2002; …”

“…For LLNA, we took LLNA thresholds derived from EC3 values for LLNA-positive chemicals and set maximum concentrations for LLNA-negative chemicals as benchmarks. For cytotoxicity, we used published CV75 values (concentration giving around 75% cell viability) (Ashikaga et al, 2010). For h-CLAT, we used threshold values of CD86 and CD54, i.e., EC150 value (estimated concentration giving a relative fluorescence intensity (RFI) = 150% for CD86) and EC200 value (estimated concentration giving a relative fluorescence intensity (RFI) = 200% for CD54) (Nukada et al, 2012).…”

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment

“…A test substance is considered a skin sensitiser in case the RFI of either CD86 or CD54 reaches defined thresholds (CD86 P 150% and/or CD54 P 200%), in at least two of three independent measurements at any concentration. Concentrations exceeding 50% cytotoxicity, measured with propidium iodide (PI), are excluded from analysis (Ashikaga et al, 2010).…”

Perspectives and strategies of alternative methods used in the risk assessment of personal care products

“…This method differs from MUSST by the cell line used (THP1, monocytes leukemia cell line) and by the measurement of one more marker: CD54 [60]. More substances have been evaluated than for MUSST, 117, and compared with LLNA sensitivity is 88%, specificity 75% and accuracy 85% [61].…”