The modified IL-8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false-negative judgment of lipophilic sensitizers with logK ow values > 3.5

Yutaka Kimura, Chizu Fujimura, Setsuya Aiba

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)749-758
Number of pages10
JournalArchives of Toxicology
Volume95
Issue number2
DOIs
Publication statusPublished - 2021 Feb

Keywords

  • Lipophilic chemicals
  • Luciferase assay
  • Reporter assay
  • Skin sensitisation test

ASJC Scopus subject areas

  • Toxicology
  • Health, Toxicology and Mutagenesis

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