Abstract
False-negative judgment due to poor chemical solubility is a problem with in vitro skin sensitisation tests. Water-insoluble chemicals are typically dissolved in DMSO in most sensitisation tests but precipitate when diluted with medium beyond their solubility in water. Such tests lack procedures to rule out false-negative judgments due to poor solubility. The IL-8 Luc assay (OECD442E) is unique in that if chemicals do not dissolve at 20 mg/mL in medium and have no effect on IL-8 luciferase activity (IL8LA), they are classified as indeterminate. The purpose of the present study was to reduce the number of indeterminate chemicals and improve assay performance. The IL-8 Luc assay can simultaneously examine glyceraldehyde 3-phosphate dehydrogenase luciferase activity (GAPLA) and IL8LA, and thus we examined the correlation between the reduction of GAPLA (defined as Inh-GAPLA) and the reduction of propidium iodide (PI)-excluding cells for three sensitizers and three non-sensitizers. We observed a significant correlation between luciferase activity driven by the GAPDH promoter of THP-G8 cells and the number of viable cells. Furthermore, chemicals providing an Inh-GAPLA value below 0.8 always reduced the ratio of PI-excluding cells to less than 0.6. Using the modified criteria, indeterminate chemicals are judged as negative if they provide Inh-GAPLA values below 0.8. This modification reduced the number of indeterminate chemicals and increased specificity, highlighting the unique advantage of the IL-8 Luc assay.
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This study was supported by Grants-in-Aid from the Ministry of Economy, Trade and Industry (METI), Japan, the Ministry of Health, Labor and Welfare (MHLW), Japan, and the Japanese Society for Alternatives to Animal Experiments (JSAAE), Japan.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by CF, YK and SA. The first draft of the manuscript was written by SA and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Kimura, Y., Fujimura, C. & Aiba, S. The modified IL-8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false-negative judgment of lipophilic sensitizers with logKow values > 3.5. Arch Toxicol 95, 749–758 (2021). https://doi.org/10.1007/s00204-020-02934-9
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DOI: https://doi.org/10.1007/s00204-020-02934-9