Extending the applicability domain of the human cell line activation test (h-CLAT).

ALTEX Pub Date : 2021-01-01 Epub Date: 2020-08-04 DOI:10.14573/altex.2001242
Karsten R Mewes, Ursula Engels, Birgit Eicker, Dirk Petersohn
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引用次数: 2

Abstract

Cosmetic ingredients must be toxicologically assessed to determine their skin sensitizing potential. The in vitro human cell line activation test (h-CLAT; OECD TG 442E) addresses the activation of dermal dendritic cells by analyzing specific protein expression after exposure of THP-1 cells to the test chemical. According to the protocol, FITC-labeled antibodies are used for protein detection. However, some chemicals show strong autofluorescence at FITC-specific wavelengths so that antibody-specific signals cannot be distinguished appropriately from autofluorescence background. This leads to inconclusive or false-negative predictions. Alternative fluorochromes can be used if their equivalence with the FITC-labeled antibodies is proven. In the current paper we describe the results of a proficiency exercise, based on the proficiency chemicals listed in the guideline, with FITC-labeled antibodies as the benchmark and APC-labeled anti­bodies as an alternative detection system. APC emits fluorescence at longer wavelengths, thus avoiding interference in the FITC spectrum. Irrespective of the employed fluorochrome, all chemicals were classified correctly, and the EC150 and 200 values were in the same order of magnitude. Hence, the equivalence in performance of FITC- and APC-labeled antibodies was demonstrated, and the respective demand of the guideline was fulfilled. In a case study, we then tested a proprietary oxidative hair dye using both fluorochromes. Using APC-labeled antibodies, the hair dye was unambiguously identified as a sensitizer, whereas no classification could be made with the FITC-labeled antibodies. With APC, fluorescence interference can be circumvented and the applicability domain of the h-CLAT extended to include autofluorescent chemicals.

扩展了人类细胞系激活试验(h-CLAT)的适用范围。
化妆品成分必须进行毒理学评估,以确定其皮肤致敏潜力。体外人细胞系激活试验(h-CLAT);OECD TG 442E)通过分析THP-1细胞暴露于测试化学物质后的特定蛋白表达来解决真皮树突状细胞的活化问题。根据方案,fitc标记的抗体用于蛋白质检测。然而,一些化学物质在fitc特异性波长表现出强烈的自身荧光,因此抗体特异性信号无法从自身荧光背景中适当区分出来。这导致了不确定或假阴性的预测。如果证明替代荧光染料与fitc标记的抗体等效,则可以使用替代荧光染料。在本文中,我们描述了基于指南中列出的熟练化学品的熟练程度练习的结果,以fitc标记的抗体为基准,apc标记的抗体作为替代检测系统。APC发出的荧光波长较长,从而避免了对FITC光谱的干扰。无论使用何种荧光染料,所有化学品都被正确分类,EC150和200值处于同一数量级。因此,FITC标记抗体和apc标记抗体的性能是相等的,满足了指南的各自要求。在一个案例研究中,我们测试了一种使用两种荧光染料的专有氧化染发剂。使用apc标记的抗体,可以明确地将染发剂确定为致敏剂,而使用fitc标记的抗体则无法进行分类。利用APC,可以规避荧光干扰,并将h-CLAT的适用范围扩展到包括自荧光化学物质。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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