Nelissen Inge, Haase Andrea, Anguissola Sergio, Rocks Louise, Jacobs An, Willems Hanny, Riebeling Christian, Luch Andreas, Piret Jean-Pascal, Toussaint Olivier, Trouiller Bénédicte, Lacroix Ghislaine, Gutleb Arno C, Contal Servane, Diabaté Silvia, Weiss Carsten, Lozano-Fernández Tamara, González-Fernández África, Dusinska Maria, Huk Anna, Stone Vicki, Kanase Nilesh, Nocuń Marek, Stępnik Maciej, Meschini Stefania, Ammendolia Maria Grazia, Lewinski Nastassja, Riediker Michael, Venturini Marco, Benetti Federico, Topinka Jan, Brzicova Tana, Milani Silvia, Rädler Joachim, Salvati Anna, Dawson Kenneth A
Health Department, Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium.
Department of Chemicals and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.
Nanomaterials (Basel). 2020 Jul 22;10(8):1430. doi: 10.3390/nano10081430.
The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.
纳米安全研究的质量和相关性构成了重大挑战，以确保其作为可持续创新及后续竞争经济优势的支持工具的关键作用。然而，在过去几年中，明显相互矛盾和无定论的研究结果数量有所增加，这表明有必要在纳米安全研究领域引入统一的方案和良好规范。因此，我们旨在评估在15个欧洲实验室中，针对纳米材料细胞毒性评估的3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四氮唑（MTS）检测进行最佳实践培训和实验室间比较（ILC）是否能提高纳米安全测试的质量。我们使用了两种描述清晰的模型纳米颗粒，40纳米的羧基化聚苯乙烯（PS-COOH）和50纳米的氨基修饰聚苯乙烯（PS-NH2）。我们采用分层方法，使用完善的标准操作规程（SOP），并共享相同的细胞、血清和纳米颗粒。我们首先测定细胞生长速率（第1层），随后进入方法转移阶段，在此阶段所有实验室对MTS检测进行首次ILC（第2层）。根据第2层的结果和实验室实践调查，组织了特定培训，并完善了MTS检测SOP。这使得第3层的实验室内和实验室间重现性有了很大提高。此外，我们证实PS-COOH和PS-NH2分别是合适的阴性和阳性对照纳米颗粒，可用于使用MTS检测评估纳米材料对细胞活力的影响。总体而言，我们证明了这里遵循的分层过程，结合使用SOP和代表性对照纳米材料，是必要的，并且能够实现良好的实验室间重现性，从而获得高质量的纳米毒理学数据。
