药物神经发育毒性比较及筛查模型建立

目的：为建立药物神经发育毒性筛查模型选择可评价药物毒性的指标。方法：以受精后6 h（6 hpf）的斑马鱼胚胎为实验模型，E₃培养液为溶剂对照组，不同药物不同浓度的暴露液为染毒组。计算受精后24，72，96，110 h的死亡率；受精后48，54，60，72，96 h的孵化率；观察受精后6 d（6 dpf）的幼鱼对强光刺激的惊恐逃避反射、黑暗状态下的运动行为及趋触性。结果：对6 hpf的斑马鱼胚胎染毒，氯丙嗪质量浓度>4.0 mg·L^-1、丙戊酸钠质量浓度>2.0 mg·L^-1可引起斑马鱼胚胎出现浓度依赖性死亡。氯丙嗪染毒时，与对照组、0.25 mg·L^-1染毒组相比，高浓度染毒组斑马鱼幼鱼总移动距离和平均速度均降低，且具有显著性差异（P<0.05）；药物丙戊酸钠染毒时，与对照组相比，随浓度增加呈现移动距离和平均速度先增后降趋势，高浓度组2.0 mg·L^-1移动距离和平均速度明显降低，且具有显著性差异（P<0.05）。氯丙嗪和丙戊酸钠染毒时，中间1 min光照期与前3 min钟黑暗期相比，运动平均速度均有所下降，具有显著性差异（P<0.05）；后3 min黑暗期与中间1 min光照期相比，运动平均速度均有所上升，具有显著性差异（P<0.05），且丙戊酸钠染毒时各组后3 min黑暗期运动平均速度均大于前3 min黑暗期。结论：药物氯丙嗪和丙戊酸钠可能导致斑马鱼胚胎发育障碍以及幼鱼神经行为异常，且药物丙戊酸钠毒性大于氯丙嗪。初步认为模式动物斑马鱼作为一种高通量筛选模型，其死亡率、孵化率、半数致死浓度LC₅₀、斑马鱼幼鱼总移动距离和平均速度可作为评价药物神经毒性的指标。     

核酸染料Goodview、Gelred和Gelsafe对斑马鱼胚胎发育的毒性作用

目的研究核酸染料Goodview、Gelred和Gelsafe对斑马鱼胚胎发育的影响。方法将受精后时间（hpf）为3hpf的斑马鱼胚胎随机分组,分别加入0、0.01%、0.02%、0.04%、0.08%、0.16%和0.32%浓度的Goodview、Gelred和Gelsafe 3种核酸染料,观察3种核酸染料对斑马鱼胚胎发育的影响,记录胚胎48 hpf心率,计算72和96 hpf孵化率,96 hpf死亡率及畸形率;同时观察了4、24和48 hpf 3种染料穿透胚胎膜的情况。结果 Goodview、Gelred和Gelsafe 3种核酸染料半数致死浓度（LC50）分别为0.037%、0.092%和0.130%。3种核酸染料0.04%～0.32%浓度组的胚胎48 hpf心率、72和96 hpf孵化率、96hpf死亡率和畸形率与对照组相比都存在显著差异,3种核酸染料96 hpf死亡率、72和96 hpf孵化率还存在剂量-效应关系。Gelsafe 0.08%～0.32%浓度组部分幼鱼出现心囊水肿和脊柱弯曲,0.32%Gelred组染毒后还出现眼点发育不全,Goodview0.04～0.16%浓度组部分幼鱼除心囊水肿和脊柱弯曲外,还出现尾部发育不全等畸形表征。0.01%Goodview 4 hpf能穿透胚胎膜进入胚胎内,随着浓度和时间增加,胚胎内荧光增强。而Gelred和Gelsafe 0.32%浓度下4 hpf未发现明显荧光蓄积点,24hpf和48 hpf仍未发现。结论 3种核酸染料对斑马鱼胚胎发育有毒性,随着使用浓度的增加毒性增加。     

冰片对斑马鱼胚胎发育的安全性评价

目的:研究冰片对斑马鱼胚胎发育的影响。方法:以斑马鱼胚胎为模型,二甲基亚砜为助溶剂,制备冰片溶液(33.6、67.1、134.3、268.5、537.0、1 074.0μmol/L,即冰片①、②、③、④、⑤、⑥组),并且设空白对照组,以受精后小时(hpf)为时间段,对3hpf胚胎进行暴露染毒处理,每24 h更换1次冰片溶液,分别在8、24、48、72、96 hpf时间点以显微镜观察班马鱼胚胎发育形态,测定24 hpf斑马鱼胚胎自主抽动次数、48 hpf心率、孵化率、畸形率和死亡率。实时荧光定量聚合酶链反应法检测斑马鱼胚胎Sepn1基因表达的情况。结果:与空白对照组比较,冰片④、⑤、⑥组斑马鱼胚胎48 hpf出现脊柱弯曲、心囊水肿、卵黄囊水肿,游泳异常的毒性症状;冰片②、③、④、⑤、⑥组斑马鱼胚胎24 hpf自动抽动次数减少;冰片④、⑤、⑥组斑马鱼胚胎24 hpf心率减少;在48 hpf时,冰片①、②、③、④、⑤、⑥组斑马鱼胚胎孵化率降低;在72 hpf时,冰片②、③、④、⑤、⑥组斑马鱼胚胎孵化率降低;在96 hpf时,冰片⑤、⑥组斑马鱼胚胎孵化率降低;冰片④、⑤、⑥组斑马鱼胚胎96 hpf畸形率增加;24 hpf后,冰片④、⑤、⑥组斑马鱼胚胎死亡率降低;冰片④组Sepn1 mRNA表达增强,差异有统计学意义(P<0.01或P<0.05)。结论:高剂量冰片对斑马鱼胚胎发育有明显影响,且呈现一定的时间依赖性。建议婴幼儿、孕妇和哺乳期的妇女长时间或大剂量使用含冰片的药物均应谨慎。     

斑马鱼胚胎评价5种药物的发育毒性与模型验证

目的用斑马鱼胚胎探究环磷酰胺、乙酰水杨酸、盐酸四环素、乙酸地塞米松、阿扎胞苷5种已知对人类胚胎致畸药物的毒性和安全性。方法挑选4 hpf发育正常的受精卵,采用水浴染毒法,将药物添加到人工海水中,每种药物分别设置5个浓度组,另设空白对照组和溶剂对照组,观察给药120 hpf后斑马鱼的死亡情况,统计各实验组斑马鱼胚胎的死亡数、畸形数,并求出120 hpf时斑马鱼胚胎的死亡率、畸形率、半数致死浓度(LC50)、半数致畸浓度(EC50)、致畸指数(TI)。并利用公式:TI=LC50/EC50计算出阳性药物的致畸指数。根据已经测得的LC50,求出各药物的最大非致死浓度(MNLC),分别设置1/10 MNLC、1/3 MNLC,MNLC和LC104个浓度,以沙利度胺为阳性对照,维生素C为阴性对照,人工海水为空白对照,0.5%DMSO为溶剂对照,28.5℃下作用至120 h,每天观察胚胎的发育情况,统计胚胎死亡及畸形状态。结果 5种药物的LC50从大到小依次为:环磷酰胺>阿扎胞苷>盐酸四环素>乙酰水杨酸>乙酸地塞米松。EC50从大到小依次为:环磷酰胺>盐酸四环素>阿扎胞苷>乙酰水杨酸>乙酸地塞米松。环磷酰胺、乙酰水杨酸、盐酸四环素、乙酸地塞米松、阿扎胞苷TI值分别为1.92,1.11,1.05,1.44,2.99。结论斑马鱼胚胎模型可用于初步评价药物的发育毒性和安全性。     

四溴双酚A对斑马鱼胚胎发育毒性和神经毒性研究

目的 研究四溴双酚A (Tetrabromobisphenol A,TBBPA)对斑马鱼胚胎的发育毒性和神经毒性.方法 采用斑马鱼胚胎模型,利用胚胎暴露实验分析剂量效应和畸形表观等发育毒性指标;利用胚胎自主运动、接触反应和仔鱼游泳运动,分析神经毒性指标.结果 TBBPA对斑马鱼胚胎的发育具有中度毒性,在受精后48 h(Hours Post Fertilization,hpf)的半数致死量(Concentration that led t0 50％ mortality,LC50)和半数致畸量(Concentration that led t0 50％ malformations,EC50)分别是7.9和4.7 μmol·L-1,以及在120 hpf的LC50和EC50分别是5.3和1.9 μmol·L-1.发育毒性效应具体表现为(1)在48,60,72,96 hpf均表现为低浓度促进孵化,高浓度抑制孵化;(2)引起胚胎发生尾部弯曲、未吸收卵黄囊、心包囊肿、卵黄囊水肿、游囊关闭等畸形现象;(3)胚胎畸形率和死亡率均具有剂量依赖效应,即与暴露浓度成正比.TBBPA对斑马鱼的神经毒性表现为(1)增加胚胎在19～26 hpf的自主运动频率;(2)降低胚胎在27,36,48 hpf时的接触反应能力;(3)降低胚胎在120 hpf的行为运动速度.结论TBBPA对斑马鱼胚胎具有发育毒性和神经行为毒性.     

β-氯氰菊酯对斑马鱼胚胎的发育毒性

目的以斑马鱼胚胎为模型,探讨一种高效氯氰菊酯β-氯氰菊酯对胚胎发育的影响。方法 丙酮为助溶剂,配制β-氯氰菊酯0.05,0.1,0.15,0.2,0.6和1 mg.L-1,采用换水式每12 h更换一半β-氯氰菊酯溶液,对斑马鱼胚胎进行96 h暴露处理,采用显微镜观察β-氯氰菊酯0.05,0.1,0.15,0.2,0.6和1 mg.L-1对斑马鱼胚胎发育形态,测定受精后24 h(24 hpf)自主抽动次数、48 hpf心率及孵化率、72和96 hpf体轴弯曲个体比例等。结果 与正常对照组比较,β-氯氰菊酯0.05,0.1,0.15,0.2,0.6和1 mg.L-1组斑马鱼胚胎在24 hpf前形态上未出现明显异常,48 hpf以后表现出体轴弯曲、心包囊肿等不同程度的毒性反应症状,β-氯氰菊酯0.2 mg.L-1组幼鱼胸鳍发育即受到严重抑制且黑色素减少体色偏黄;随着β-氯氰菊酯浓度的增加,斑马鱼胚胎在24 hpf时每分钟自主抽动次数由正常对照组的(0.72±0.19)次增加至(3.83±1.07)次(P<0.05);48 hpf孵化率由对照组的(15.5±4.3)%升高至(98.9±1.2)%(P<0.05)。β-氯氰菊酯0.05 mg.L-1组72 hpf和96 hpf体轴弯曲个体比例分别为6.6%和10%,β-氯氰菊酯1 mg.L-1组分别为97.8%和100%。结论 β-氯氰菊酯对斑马鱼胚胎的神经及形态发育均有明显抑制作用,并且呈现一定的时间剂量依赖性。     

阿司咪唑对斑马鱼心脏毒性的初步研究

目的 研究阿司咪唑对斑马鱼的心脏毒性,为斑马鱼的心脏毒性模型提供依据.方法 选择1、4、10、20 μmol·L-1的阿司咪唑观察其对72 hpf( hour post fertilization)斑马鱼的形态影响,包括畸形、心囊水肿、血细胞在心区堆积等情况,计算心率、死亡率及EC50,测量静脉窦-动脉球(SV-BA)间距,以及对2 hpf斑马鱼心脏发育的影响.结果 阿司咪唑处理后,斑马鱼出现出心率下降、SV-BA间距增大、血细胞在心区堆积、全身无血流、心囊水肿、心脏停止跳动等中毒现象,并具有时间、剂量依赖性;同时观察到阿司咪唑对2 hpf斑马鱼的心脏发育有明显影响.结论 利用阳性药物阿司咪唑探讨药物对斑马鱼心脏功能和发育方面的毒性,有助于建立基于斑马鱼的心脏毒性评价筛选体系.     

牛黄镇惊丸对斑马鱼胚胎发育的影响

目的 研究牛黄镇惊丸对斑马鱼胚胎发育的影响。方法 以斑马鱼胚胎为实验对象,体视光学显微镜下观察牛黄镇惊丸不同给药浓度（40、200、400、800、1 200 μg/mL）时斑马鱼胚胎的发育情况,包括胚胎致畸、致死检测。结果 给药浓度不高于200 μg/mL时,未见胚胎发育异常。浓度高于200 μg/mL（约相当于成人用量30倍,远超出正常有效剂量）时对胚胎发育具有一定的毒性,胚胎出现头和眼发育较小,瞳孔无色;心脏畸形、心率较弱、血池充血-回心血流不畅;腹部透明度较差、尾干扭曲,卵黄囊延伸结构变粗,脊索变细,无正常胚动,体长变短;撤药后畸形表型未见恢复,且毒性呈浓度相关性;半数致畸浓度（TD₅₀,受精后3 d）约373 μg/mL,半数致死浓度（LD₅₀）约为485 μg/mL。浓度为1 200 μg/mL时,胚胎在受精后1 d之内全部死亡。结论 牛黄镇惊丸给药浓度大于200 μg/mL时影响斑马鱼胚胎发育。其毒性作用发生在胚胎器官形成期,主要对胚胎的神经系统、心血管系统和骨骼等发育有影响。     

骨刺片对斑马鱼胚胎发育的急性毒性研究

目的 研究两批骨刺片样品对斑马鱼胚胎发育的急性毒性作用。方法 以斑马鱼胚胎为试验对象,观察骨刺片样品A（0.01、0.05、0.10、0.20、0.30、0.50、1.00 mg/mL）、样品B（0.01、0.05、0.10、0.50、1.00、2.00、10.00 mg/mL）对斑马鱼胚胎发育的影响,包括胚胎致畸、致死检测;同时采用超高效液相色谱法-四极杆飞行时间-质谱（UPLC-QTOF-MS）对两批样品所含化学成分进行定性分析。结果 两批样品对斑马鱼胚胎发育的影响相似,主要表现为高浓度下的致死作用,低浓度下主要以发育滞后表型为主;但二者作用浓度存在一定差异,样品A的半数致畸剂量（TD₅₀）为225.4 mg/mL,半数致死剂量（LD₅₀）为292.0 mg/mL;样品B的TD₅₀为60.3 mg/mL,LD₅₀为1.382 mg/mL。UPLC-QTOF-MS分析结果显示,两批样品所含主要化学成分基本一致,但某些成分含量存在一定差异;初步分析显示既是有效成分又是毒性成分的士的宁含量与斑马鱼胚胎试验结果呈正相关。结论 斑马鱼胚胎模型可用于评价骨刺片样品的急性毒性,两批骨刺片样品对斑马鱼胚胎发育影响均表现为高浓度下的致死作用,低浓度下,特别是样品B表现为轻度非正常表型,且均可逆。     

FABP3过表达对斑马鱼胚胎心脏发育及线粒体DNA拷贝数的影响

目的探讨脂肪酸结合蛋白(FABP)3过表达对斑马鱼胚胎死亡率、心脏发育形态及线粒体DNA拷贝数影响。方法构建FABP3过表达载体,在斑马鱼受精卵的单细胞期进行胚胎显微注射(A组);以野生型斑马鱼胚胎作为空白对照(C组)。收集受精后12、24、48、72hpf斑马鱼胚胎,利用体式显微镜观察其发育情况,并统计其死亡率;RT-PCR技术检测胚胎线粒体DNA拷贝数。结果 A组斑马鱼心脏在12、24、48、72hpf各时间点发育大体形态、死亡率与C组比较均无明显差异。A组24hpf时斑马鱼线粒体DNA的拷贝数高于C组(P<0.05);而在48、72hpf时,两组间斑马鱼线粒体DNA拷贝数无统计学差异(P>0.05)。结论 FABP3过表达对斑马鱼胚胎死亡率、心脏发育的形态无明显影响,对线粒体功能起保护作用。     

Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo

We developed and optimized a screening procedure, in which zebrafish embryos were explored as a model for the evaluation of the specific embryotoxic and teratogenic potential of chemicals. A selection of known positive (retinoic acid, valproic acid, caffeine, lithium chloride) and negative (glucose, saccharin) compounds for developmental toxicity were used to evaluate this method. We exposed embryos and evaluated embryotoxicity and morphological characteristics of the embryos at 24, 48, 72 and 144 h post fertilization. After evaluation of the induced effects, concentration–response curves were created for both embryotoxicity and teratogenic effects. Values for teratogenic indices (TI) were calculated as the ratio LC₅₀/EC₅₀. The results obtained were compared to existing data from studies with laboratory animals and humans. We demonstrated that our classification of the compounds, based on TI values, allows to distinguish teratogens from non-teratogens and supports the application of zebrafish embryos as an alternative method for developmental toxicity studies to predict effects in mammals.     

Developmental toxicity,EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

Dioxin‐like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L^?1 from 3‐h post‐fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non‐inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126‐induced developmental toxicity, we conducted ethoxyresorufin‐O‐deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real‐time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L^?1 concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L^?1 doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L^?1 at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose‐dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 201–210, 2016.     

甘脯酰阿霉素对斑马鱼胚胎的毒性评估

目的:考察甘脯酰阿霉素(Z-GP-Dox)对斑马鱼胚胎的急性毒性、心脏毒性及外周血液毒性。方法:以24hpf(受精后小时)斑马鱼胚胎为模型,二甲基亚砜(DMSO)为溶剂,以1.25、2.5、5、10、15、20μmol·L-1Z-GP-Dox与阿霉素(Dox)对48、72、96hpf胚胎存活率的影响考察急性毒性,以5、10μmol·L-1Z-GP-Dox与Dox对72hpf胚胎心率的影响考察心脏毒性,以对红细胞和嗜中性细胞数量的影响考察外周血液毒性,并设溶剂对照组进行比较。结果:溶剂对胚胎存活无明显影响,Dox>10μmol·L-1时,96hpf胚胎存活率小于25%,而Z-GP-Dox为20μmol·L-1时依然有70%以上的胚胎存活;与溶剂对照组比较,5、10μmol·L-1Dox明显降低胚胎的心率和嗜中性细胞数量,而5、10μmol·L-1Z-GP-Dox对胚胎心率、红细胞和嗜中性细胞数量均无明显影响。结论:Z-GP-Dox的毒性比Dox低。     

瑞舒伐他汀钙通过下调血管内皮生长因子的表达抑制斑马鱼血管发育

目的:通过研究瑞舒伐他汀钙对斑马鱼血管发育的影响,探讨瑞舒伐他汀钙抑制血管的作用机制。方法:将瑞舒伐他汀钙处理的斑马鱼胚胎作为实验组,以0.5%二甲亚砜处理的胚胎作为对照组。从受精卵形成后0.5h开始刺激,受精后24h观察药物对斑马鱼血管发育的影响。使用荧光显微镜观察新生血管状态,记录血管缺失数目,分别从受精后0、6、12和24h药物处理后的斑马鱼胚胎中提取总的RNAs,采用实时定量逆转录聚合酶链反应(RT-PCR)检测血管内皮生长因子A(vascular endothelial growth factor A,VEGF-A)的表达变化。结果:瑞舒伐他汀钙可明显抑制斑马鱼血管发育,且在0.01～0.08nmol/L浓度范围内抑制作用与药物浓度成正比。实验组较对照组VEGF-A表达量降低,在药物作用的24h具有显著性差异(P<0.05)。结论:瑞舒伐他汀钙可通过减少VEGF-A的表达抑制斑马鱼血管发育。     

Early life stage and genetic toxicity of stannous chloride on zebrafish embryos and adults: Toxic effects of tin on zebrafish

Humans are exposed to stannous chloride (SnCl₂), known as tin chloride, present in packaged food, soft drinks, biocides, dentifrices, etc. Health effects in children exposed to tin and tin compounds have not been investigated yet. Therefore, we evaluated the possible teratogenic effects and genotoxic of SnCl₂ in zebrafish (Danio rerio) adults and their embryos. In the embryo–larval study, SnCl₂ showed embryo toxicity and developmental delay after exposure to the various concentrations of 10–250 μM for 120 h. Teratogenic effects including morphological malformations of the embryos and larvae were observed. The embryos exposed to 100 μM displayed tail deformation at 28 hpf and the larvae exposed to 50 μM showed reduced body growth, smaller head and eyes, bent trunk, mild pericardial edema, and smaller caudal fin at 96 hpf. The results of the teratological study show that SnCl₂ induced a significant decrease in the number of living embryos and larvae. Regarding the chromosome analysis, SnCl₂ induced a dose‐dependent increase in the micronucleus (MN) frequency in peripheral erythrocytes of adult zebrafish. In blood cells, the 25 μM dose of SnCl₂ caused a nonsignificant increase in the total chromosomal aberrations, but the high doses significantly increased the total number of chromosomal aberrations compared with the control groups. Overall, the results clearly indicate that SnCl₂ is teratogenic and genotoxic to zebrafish. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

Toxic effects of celastrol on embryonic development of zebrafish (Danio rerio)

Celastrol is a terpenoid purified from Tripterygium wilfordii Hook F. As a natural product with pharmacological activities, this compound is a promising candidate for drug development. To provide more information about its toxicity for clinical trials, toxicity assessment of celastrol was conducted with zebrafish model in vivo. 1hour post-fertilization (hpf) embryos were treated with various concentrations of celastrol for 120h. Developmental phenotypes were observed and survival rates were recorded. The results showed that the hatching rates of embryos treated with 1.0μM or higher celastrol were significantly lower. Embryos exposed to 1.0μM celastrol had no blood flow in trunk vessels at 48hpf with a median effect concentration (EC(50)) of 0.94μM. At 72hpf serious edema in pericardial sac was observed in the surviving larvae (hatched from embryos treated with 1.5μM celastrol). Bent tails or hook-like tails were seen as 0.5μM celastrol and the EC(50) for tail malformation was 0.66 μM at 72hpf. The lethal effect of celastrol on zebrafish embryos was dose-dependent and the LC(50) values of celastrol on 1hpf embryos were approximately 1.40μM. These results indicate that celastrol affects the normal development of zebrafish embryo in μM concentrations.     

Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds

To anticipate to increased testing needs for reproductive toxicity and 3R approaches, we studied zebrafish embryo/larva as an alternative for animal testing for developmental toxicity and embryotoxicity and evaluated a training set of 27 compounds with a standardized protocol. The classification of compounds in the zebrafish embryo/larva assay, based on a prediction model using a TI (teratogenic index) cut-off value of 2, was compared to available animal and human data. When comparing the classification of compounds in the zebrafish embryo/larva assay to available animal classification, a sensitivity of 72% and specificity of 100% were obtained. The predictive values obtained in comparison to a limited set of human data were 50, 60% respectively for teratogens, non-teratogens. Overall, we demonstrated that the zebrafish embryo/larva assay, may be used as screening tool for prioritization of compounds and could contribute to reduction of animal experiments in the field of developmental toxicology.