鱼类急性毒性体外替代标准试验方法的应用与适用性评价
Application of Standard in vitro Alternative Testing Method for Acute Fish Toxicity and Evaluation on Its Applicability
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摘要: 经济合作与发展组织(Organization for Economic Co-operation and Development, OECD)2021年颁布虹鳟鱼鳃上皮细胞系RTgill-W1急性毒性试验标准方法(OECD 249,2021)作为鱼类急性毒性试验或其预试验的体外替代方法,因试验体系小、通量高、周期短而具良好的应用潜能。但方法被认为对神经毒物可能存在预测偏差,尚无对金属化合物的应用报道。此外,细胞毒性数据与鱼体内数据的相关性未知。目前尚无采用该标准方法的实际应用报道,故方法实际适用性不明确。因此,采用涵盖水生毒性试验常用参比物、金属化合物、神经毒物在内的10种化学品进行该方法应用评价研究。结果表明,参照标准方法,同一实验室结果具可重复性及再现性。除通过细胞凋亡途径导致毒性效应的多菌灵外,其余化学品细胞急性毒性EC50值与标准鱼种(OECD 203推荐鱼种及稀有鮈鲫)急性毒性LC50值呈良好线性关系(相关系数(r2)为0.7009~0.7975)。细胞急性毒性EC50与鱼类急性毒性LC50存在数值差异(差异在10倍以内),但同一实验室采用标准方法建立化学品细胞毒性与标准鱼种的体内急性毒性良好转换关系后,可通过细胞急性毒性准确预测鱼体内急性毒性。此外,OECD 249方法可拓展用于评估高分子量、难溶化学品是否可透过生物膜并产生毒性,决定其登记申报时是否可豁免水生毒性试验。Abstract: As an alternative method of acute fish toxicity test or its range-finding test, a standard in vitro testing method by using gill epithelial cell lines of rainbow trout (OECD 249, 2021) has been published by Organization for Economic Co-operation and Development (OECD) in 2021. Due to the advantages of small test system, high throughput and short test period, this standard method showed good potential for application. However, it also has its limitations, e.g. maybe having predictive bias for neurotoxicants, and having no application for metal compounds. In addition, the correlation of cytotoxicity and acute fish toxicity, as well as its applicability in practical application is still unclear. Therefore, ten test chemicals including reference substance commonly used in aquatic toxicity test, metal compounds and neurotoxic substances have been used in this study to evaluate the applicability of OECD 249. The results indicated that RTgill-W1 cytotoxicity tests are repeatable and reproducible by using standard testing method (i.e. OECD 249) in the same laboratory. With the exception of carbendazim, which leads toxic effects in fish through the apoptotic pathway, there is a good linear relationship between the EC50 values of acute cytotoxicity of other chemicals and the LC50 values of acute toxicity by using standard fish species (i.e. test species recommended by OECD 203 and Gobiocypris rarus) (with the correlation coefficient (r2) of 0.7009 to 0.7975). Although there were certain differences between EC50 values of cytotoxicity and LC50 values of acute fish toxicity (the difference was less than 10 folds), a good conversation relationship between cytotoxicity and in vivo acute toxicity by using standard fish species could still be established in the same laboratory, which means accurate prediction of fish LC50 maybe achieved by using EC50 of cytotoxicity. Furthermore, OECD 249 testing method can be also used to assess the possibility of toxicity produced by high molecular chemicals with limited water solubility and passing through biomembranes, and then determine whether in vivo aquatic toxicity tests can be waived for chemical registration.
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Key words:
- fish /
- acute toxicity /
- in vitro alternative test /
- RTgill-W1
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