MatTek Corporation, Ashland, MA 01721, USA.
Altern Lab Anim. 2009 Dec;37(6):671-89. doi: 10.1177/026119290903700611.
A skin irritation test (SIT) utilising a common protocol for two in vitro reconstructed human epidermal (RhE) models, EPISKIN and EpiDerm, was developed, optimised and evaluated as a replacement for the in vivo rabbit skin irritation test in an ECVAM-sponsored validation study. In 2007, both RhE models were recognised by an independent peer-review panel and the ECVAM Scientific Advisory Committee (ESAC) as validated for use with the common SIT protocol. The EPISKIN SIT was endorsed as a full replacement of the in vivo rabbit test. Since the EpiDerm SIT proved to be less sensitive than the in vivo test and the EPISKIN SIT, the test was recognised as a validated component of a tiered testing strategy, in which positive results are accepted and negative results require further confirmation. The ESAC, in its April 2007 statement, also recommended increasing the sensitivity of the EpiDerm SIT, in order to gain the full acceptance. Analysis of the EpiDerm and EPISKIN data from the ECVAM validation study indicated that the lower sensitivity of the EpiDerm SIT might be linked to the more robust barrier properties of the EpiDerm model. This hypothesis was also in line with results published previously. To overcome the relatively low sensitivity of the EpiDerm protocol as a hindrance to full regulatory acceptance, a modification of exposure conditions was introduced into the protocol to achieve better agreement with the in vivo rabbit data. In the Modified EpiDerm SIT protocol, the test chemical exposure time was increased from 15 minutes to 60 minutes. In addition, part of the exposure was performed at 37 degrees C. When the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay endpoint was used for classification, a significant increase of sensitivity was obtained (86.1%), whilst maintaining the high specificity of the method (76.3%). With the change to the EU classification system, which now uses higher cut-off for the classification of irritants, the sensitivity of the Modified EpiDerm SIT increased to above 90%. The measurement of interleukin (IL)-1alpha release did not further contribute to improvement of the method. The results demonstrate that the modified EpiDerm SIT protocol has the required sensitivity and specificity to be accepted as a stand alone method for complete replacement of the in vivo rabbit test.
开发了一种利用两种体外重建人类表皮（RhE）模型 EPISKIN 和 EpiDerm 的共同方案进行皮肤刺激试验（SIT），并在 ECVAM 赞助的验证研究中对其进行了优化和评估，以替代体内兔皮肤刺激试验。2007 年，两个 RhE 模型都得到了独立同行评审小组和 ECVAM 科学咨询委员会（ESAC）的认可，可用于共同 SIT 方案。EPISKIN SIT 被认可为体内兔试验的完全替代品。由于 EpiDerm SIT 证明不如体内试验和 EPISKIN SIT 敏感，因此该试验被认为是分层测试策略的一个验证组成部分，其中阳性结果被接受，阴性结果需要进一步确认。ESAC 在其 2007 年 4 月的声明中还建议提高 EpiDerm SIT 的灵敏度，以获得完全认可。对 ECVAM 验证研究中 EpiDerm 和 EPISKIN 数据的分析表明，EpiDerm SIT 的灵敏度较低可能与 EpiDerm 模型更强的屏障特性有关。这一假设也与之前发表的结果一致。为了克服 EpiDerm 方案作为完全监管接受的障碍的相对低灵敏度，引入了暴露条件的修改，以使其与体内兔数据更好地一致。在改良 EpiDerm SIT 方案中，测试化学暴露时间从 15 分钟增加到 60 分钟。此外，部分暴露在 37°C 下进行。当使用 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐（MTT）活力测定终点进行分类时，获得了灵敏度的显著提高（86.1%），同时保持了方法的高特异性（76.3%）。随着欧盟分类系统的改变，现在对刺激性物质的分类使用更高的截止值，改良 EpiDerm SIT 的灵敏度提高到 90%以上。白细胞介素（IL）-1alpha 释放的测量并未进一步提高方法的性能。结果表明，改良 EpiDerm SIT 方案具有所需的灵敏度和特异性，可作为替代体内兔试验的独立方法。
