The performance of an <i>in vitro</i> skin sensitisation test, IL-8 Luc assay (OECD442E), and the integrated approach with direct peptide reactive assay (DPRA)

Abstract

In all current in vitro skin sensitisation assays, DMSO is used to dissolve water-insoluble chemicals. However, our previous study suggested the superiority of the modified IL-8 Luc assay (mIL-8 Luc), in which X-VIVO TM 15 is used to dissolve chemicals, over the original assay using DMSO (oIL-8 Luc). In this study, to confirm the superiority of the mIL-8 Luc, we first increased the number of chemicals examined and demonstrated the superiority of the mIL-8 Luc, in which the mIL-8 Luc provided 87.6% of sensitivity, 74.2% of specificity, and 84.6% of accuracy. Next, to clarify the cause of false negative judgment by the mIL-8 Luc, we examined the effects of physical properties of chemicals on judgment. The results demonstrated that high molecular weight, high LogKo/w, or poor water solubility, did not cause false negative judgment. When it was accepted as an OECD test guideline, the criteria of the mIL-8 Luc to determine sensitisers were modified to further decrease false negative judgment by poor solubility. By applying the new criteria, the test guideline IL-8 Luc assay (tgIL-8 Luc) improved sensitivity but decreased specificity and increased the number of chemicals that cannot be judged. To overcome this problem, we examined a simple combination of the tgIL-8 Luc with direct peptide reactive assay (DPRA), which could improve specificity and decrease the number of the chemicals that cannot be judged. These data suggest that the tgIL-8 Luc is a promising in vitro skin sensitisation assay in combination with other in vitro or in chemico methods.

Development of a novel in vitro assay to evaluate environmental water using an IL-8 reporter cell line

“…In this study, we first demonstrated that TLR 1, 2, 4, 5, 6, NLR 1 and 2 agonists augmented IL8LA and that LPS dose-dependently stimulated IL8LA with the lowest concentration of 0.03 ng/ml or 0.4 EU/ml. In addition, we have already reported that most of haptens as well as detergents also stimulated IL8LA (Kimura et al, 2015[ 14 ], 2018[ 16 ]). Therefore, IL-8 Luc assay (H) can respond to a variety of environmental contaminants other than endotoxin, such as bacterial toxins from gram positive and negative bacteria and chemical pollutants such as haptens and detergents.…”

“…We used THP-G8 cells derived from a human acute monocytic leukemia cell line THP-1 cell line, which contained stable luciferase orange gene (SLO) regulated by IL-8 promoter and stable luciferase red gene (SLR) by G3PDH promoter (Kimura et al, 2015[ 14 ], 2018[ 16 ]; OECD, 2018[ 22 ]; Takahashi et al, 2011[ 28 ]). THP-G8 cells (5x10 4 cells/100 µL/well) in 96-well black plates (Greiner bio-one GmbH, Frickenhausen, Germany) were cultured for 6 hours with lipopolysaccharide (LPS, from E. coli 026:B6 ( > 10,000 EU/mg, Sigma, St. Louis, MO), TLR agonists (InvivoGen, San Diego, CA), NLR agonists (InvivoGen, San Diego, CA), representative haptens, methylisothiazolinone and glutaraldehyde or 10 µL of the collected environmental water autoclaved for 20 min at 121°C and 2 atm.…”

“…This cell line was established for the purpose of screening the immunotoxicity of various chemicals, such as environmental pollutants, and drugs. Indeed, the usefulness of this cell line has been already demonstrated in hazard identification of skin sensitizing chemicals (Kimura et al, 2015[ 14 ], 2018[ 16 ]; OECD, 2018[ 22 ]; Takahashi et al, 2011[ 28 ]), characterizing immunotoxic chemicals (Kimura et al, 2014[ 13 ], 2018[ 15 ]), and quantifying the immunotoxicity of Asian desert dusts (Watanabe et al, 2014[ 35 ], 2015[ 36 ]). These studies also ensure the stability of this cell line.…”

THP‐1 reference data: Proposal of an in vitro branched evolution model for cancer cell lines

“…THP‐1 is listed in the LL‐100 panel for blood cancer research 3 and is also known as a representative monocyte cell line, which differentiates into macrophages 4 . Focusing on its function as dendritic cells, THP‐1 is employed for the assessment of skin sensitization in the OECD guidelines to test chemicals 5 …”