Tetracycline hydrochloride (TH), indomethacin (IM), and bezafibrate (BF) belong to the three different important classes of pharmaceuticals, which are well known for their toxicity and environmental concerns. However, studies are still elusive to highlight the mechanistic toxicity of these pharmaceuticals and rank them using both, the toxicity prediction and confirmation approaches. Therefore, we employed the next generation toxicity testing in 21st century (TOX21) tools and estimated the in vitro/vivo toxic endpoints of mentioned pharmaceuticals, and then confirmed them using in vitro/vivo assays. We found significant resemblance in the results obtained via both approaches, especially in terms of in vivo LC50 s and developmental toxicity that ranked IM as most toxic among the studied pharmaceuticals. However, TH appeared most toxic with the lowest estimated AC50s, the highest experimental IC50s, and DNA damages in vitro. Contrarily, IM was found as congener with priority concern to activate the Pi3k-Akt-mTOR pathway in vitro at concentrations substantially lower than that of TH and BF. Further, IM exposure at lower doses (2.79–13.97 μM) depressed the pharmaceuticals detoxification phase I (CYP450 s), phase II (UGTs, SULTs), and phase III (TPs) pathways in zebrafish, whereas, at relatively higher doses, TH (2.08–33.27 μM) and BF (55.28–884.41 μM) partially activated these pathways, which ultimately caused the developmental toxicity in the following order: IM > TH > BF. In addition, we also ranked these pharmaceuticals in terms of their particular toxicity to myogenesis, hematopoiesis, and hepatogenesis in zebrafish embryos. Our results revealed that IM significantly affected myogenesis, hematopoiesis, and hepatogenesis, while TH and BF induced prominent effects on hematopoiesis via significant downregulation of associated genetic markers, such as drl, mpx, and gata2a. Overall, our findings confirmed that IM has higher toxicity than that of TH and BF, therefore, the consumption of these pharmaceuticals should be regulated in the same manner to ensure human and environmental safety.

盐酸四环素（TH），吲哚美辛（IM）和苯扎贝特（BF）属于三种不同的重要药物类别，以其毒性和对环境的关注而闻名。但是，仍然缺乏研究来突出这些药物的机械毒性，并使用毒性预测和确认方法对它们进行排名。因此，我们采用了21世纪的下一代毒性测试（TOX21）工具，并估算了所述药物的体外/体内毒性终点，然后使用体外/体内测定法对其进行了确认。我们发现通过两种方法获得的结果都非常相似，特别是在体内LC50和发育毒性将IM列为研究药物中毒性最高的一种。但是，TH的毒性最高，估计的AC50最低，实验的IC50最高，体外DNA损伤最大。相反，发现IM是优先考虑的同质物，可在体外激活Pi3k-Akt-mTOR途径浓度远低于TH和BF。此外，在较低剂量（2.79–13.97μM）下的IM暴露抑制了斑马鱼中药物的排毒阶段I（CYP450 s），阶段II（UGT，SULTs）和阶段III（TPs）途径，而在剂量相对较高时，TH （2.08–33.27μM）和BF（55.28–884.41μM）部分激活了这些途径，最终以下列顺序引起了发育毒性：IM> TH> BF。此外，我们还根据这些药物对斑马鱼胚胎中的肌发生，造血作用和肝发生的特定毒性进行了排名。我们的研究结果表明，IM显着影响肌生成，造血和肝生成，而TH和BF通过以下途径对造血产生显着影响相关遗传标记（例如drl，mpx和gata2a）的显着下调。总体而言，我们的发现证实IM的毒性高于TH和BF，因此，应以相同的方式调节这些药物的消耗量，以确保人体和环境安全。