重组人胰岛素注射液临床前生物相似性评价研究

目的 通过Beagle犬长期给药毒性试验及免疫原性试验,评价重组人胰岛素注射液(Insulin R)与原研药(Humulin R)的生物相似性。方法 进行Beagle犬scInsulin R 30 d停药14 d长期给药毒性试验,Insulin R剂量为0.5、1.0、1.5 IU/kg,Humulin R剂量为1.5 IU/kg,检测指标包括一般状况、进食量、体温、心电图、血液学、凝血、血清生化、电解质及尿液等,于给药期结束及停药期结束分两次剖杀动物,测定主要脏器的脏器系数并进行常规病理组织学检查;ELISA法检测Beagle犬血清的免疫原性。结果 Beagle犬重复sc Insulin R 30 d毒性试验中,中、高剂量组部分动物出现食欲降低、食量减少,高剂量1只雄性动物出现流涎、站立不稳、心率加速、抽搐等低血糖症状,中、高剂量组动物心率增加;原研药组与InsulinR高剂量组毒性反应相当;Beagle犬给予Insulin R 30 d未检测到抗药抗体。结论 Beagle犬sc Insulin R 30 d时的最大无毒反应剂量为0.5 IU/kg,相当于临床等效剂量的0.5倍,且未产生抗药抗体,与Humulin R具有较好的临床前生物相似性。     

甘精胰岛素注射液的临床前安全性评价

目的 通过Beagle犬长期毒性试验及其伴随的免疫原性试验,观察甘精胰岛素注射液的毒性反应,确定未观察到临床不良反应的剂量水平（NOAEL）,为临床不良反应监测及防治提供参考。方法 健康Beagle犬40条,随机分为甘精胰岛素注射液低、中和高（0.5、1.0、2.0 IU/kg）剂量组、溶媒对照组及原研对照组（来得时,2.0 IU/kg）,每组8只动物,雌雄各半。连续sc给药30天,停药恢复16天。试验期间进行一般体征观察、进食量、体质量、肛温、全血血糖及心电图检查;测定血液学常规与凝血指标、血清生化、尿液常规等指标;进行骨髓细胞形态学检查、大体剖检检查以及组织病理学检查。采用间接ELISA法检测不同时期动物血清抗药的结合抗体;采用体外生物活性HPLC法检测产生抗药抗体的阳性血清的中和活性。结果 在给药期的第8和10天,原研对照组和高剂量组各有1只雌性动物在给药后5～6 h出现抽搐和流涎等由低血糖所致的异常症状,其中高剂量组的该异常动物于次日死亡;给药期第11天的心电图检查发现高剂量组的T波倒置比例略高于溶媒对照组（5/7 vs 1/8）,停药后恢复正常;其余存活动物的体质量、肛温、进食量、尿常规、血液学、血清生化和组织病理学等均未见毒理学意义的异常改变。免疫原性结果显示,仅有高剂量组1只雄性动物于给药期第12天产生抗药抗体,抗体滴度为1:16,阳性率为14.3%,产生结合抗体的血清样品经检测为非中和活性抗体。结论 在本试验条件下,Beagle犬sc重复给予甘精胰岛素注射液的NOAEL为1.0 IU/kg,该剂量相当于临床拟用剂量的2倍。该药在高剂量下可能对个别Beagle犬具有较弱的免疫原性。     

阿哌沙班类似物SU-142口服给药4周对Beagle犬的亚慢性毒性研究

目的 研究阿哌沙班的新型结构类似物SU-142重复口服给药4周对Beagle犬产生的毒性反应。方法 40条健康Beagle犬按体质量随机分为溶媒对照组、SU-142低剂量组、SU-142中剂量组和SU-142高剂量组,每组10条,♀♂各半。低、中、高剂量组给药剂量分别为50,150,300 mg·kg^-1,溶媒对照组给予0.5% CMC-Na,给药体积均为5 mL·kg^-1。每天给药1次,共给药4周,恢复2周,期间对各项毒理学指标进行检测,并分别于首次和末次给药后1,2,4,8 h对血凝指标进行监测。结果 中剂量组1条犬首次给药后开始出现呕吐症状,至第7天恢复,未见其他异常反应。由于该症状比较轻微,且发生率低,认为是动物的个体差异导致的偶发性反应。其他动物的一般体征均未见明显异常。血凝学检测结果显示,SU-142处理组的凝血酶原时间（PT）和凝血酶时间（TT）自首次给药后1 h开始上升,给药期间显著高于溶媒对照组,至末次给药后4 h达峰,至此,高剂量组PT和TT均为溶媒对照组的约2倍,呈现明显的量效和时效关系。活化部分凝血活酶时间（APTT）给药后,均呈现剂量依赖性升高,但时效关系不明显。停药2周后,PT、TT和APTT基本恢复至基础水平。其他心电图、血液学、血液生化和病理组织学等指标均未见明显的与SU-142处理相关的改变。结论 SU-142口服给药4周对Beagle犬的主要毒性靶标为凝血系统,为SU-142药效作用的延伸和放大所致,作用可逆。本研究未发现供试品作用机制以外的脱靶毒效应。     

氢溴酸樟柳碱注射液大鼠单次和重复给药毒性试验研究

目的 通过SD大鼠的单次和重复静脉给药毒性试验,评价氢溴酸樟柳碱注射液的安全性。方法 单次给药毒性试验采用最大耐受量法,观察大鼠的死亡情况和毒性反应。重复给药毒性试验：将大鼠随机分为溶媒对照组和氢溴酸樟柳碱10、50、200 mg/kg剂量组,每组30只,尾iv给药,连续13周,停药恢复4周。进行各项毒理学指标检测。结果 急性毒性试验：氢溴酸樟柳碱注射液在364.5~504.5 mg/kg对大鼠产生明显毒性,症状有给药时尖叫、俯卧、后肢无力、颤抖、抽搐、惊厥、瞳孔散大、尾部发绀等,甚至造成个别动物死亡。重复给药毒性试验：50、200 mg/kg剂量组出现体质量增长减缓,摄食下降,给药后尖叫,鼻端、眼周异常分泌物增多,皮肤脱毛、结痂,耳廓溃疡、缺损,瞳孔散大,尾部发绀,血红蛋白（HGB）、红细胞压积（HCT）、Cl-浓度升高等症状,200 mg/kg剂量组还出现给药后肌张力减退、颤抖、抽搐、呼吸困难、皮下炎性包块等表现。溶媒对照组和200 mg/kg剂量组动物注射部位均出现静脉炎及静脉周围炎,严重程度无差异,停药4周后病变减轻。结论 氢溴酸樟柳碱注射液SD大鼠静脉单次给药的最大耐受量（MTD）为428.8 mg/kg,约相当于临床剂量的2 573倍;重复给药毒性试验未见明显毒性反应剂量（NOAEL）为10 mg/kg,约相当于临床剂量的60倍。     

4-甲基环十五烷酮单次给药毒性试验研究

目的 研究单次灌胃给予4-甲基环十五烷酮（4-methylcyclopentadecanone,4-MCPC）对Beagle犬和小鼠产生的毒性。方法 采用经典的半数致死量法（LD₅₀法）和最大给药量法分别进行小鼠和Beagle犬单次给药毒性试验,动物分组后灌胃给予不同剂量的4-甲基环十五烷酮,给药1次,给药后观察动物毒性反应,检测动物体重、死亡率、血液学、血液生化学等指标,药后14 d观察结束剖检,检查主要脏器的病变。结果 小鼠单次给予4-甲基环十五烷酮的LD₅₀为9.41 g·kg^-1,死亡鼠急性毒性症状主要为水样便、竖毛、俯卧不动、翻正反应消失继而死亡。Beagle犬单次给予4-甲基环十五烷酮的最大给药量为18.40 g·kg^-1,最大耐受量为1.56 g·kg^-1,Beagle犬毒性症状主要为稀便、呕吐、精神萎靡、厌食、活动减少等反应。临床检验结果显示18.40、12.24 g·kg^-1剂量下Beagle犬凝血酶时间（TT）明显延长,谷丙转氨酶（ALT）、谷草转氨酶（AST）、尿素氮（BUN）、肌酐（CR）显著升高。结论 4-甲基环十五烷酮对小鼠和Beagle犬的急性毒性较小,安全范围较大。     

聚乙二醇化重组人胰岛素注射液Beagle犬毒动学研究

目的 研究毒性剂量暴露下聚乙二醇化重组人胰岛素注射液（PEG-Det）的毒动学（TK）,以评价系统暴露与剂量、时间及毒性结果之间的关系,通过重复给药分析药物在体内是否存在蓄积及代谢方式是否改变等特性。方法 32只健康Beagle犬随机分为4组,每组8只,雌雄各半,分别sc低、中、高剂量（37.5、75.0、150.0 μg/kg）PEG-Det及溶媒,每周给药2次,重复给药9个月。分别于首次（d1）、中期（d89）和末期（d260）给药后采用放射免疫分析（RIA）法检测不同时间血药浓度,采用罗氏血糖仪同步测定动物血糖水平。试验数据采用DAS 3.0药动程序拟合分析并计算TK参数。结果 各剂量组动物sc给药后,随血药浓度升高伴随血糖降低,且与给药剂量呈正相关;随给药频率增加,血糖降低幅度减小;单次和多次给药后,PEG-Det的C_max和AUC与剂量均呈正相关;随给药频率增加,各剂量组的C_max和AUC_ss降低,且给药末期（9个月）的蓄积指数（R_Cmax和R_AUC）均小于1;各剂量组在给药不同阶段的消除半衰期t_1/2z为20~30 h;达峰时间T_max和清除率CL_z/F均在一定范围内波动,不与剂量相关。结论 Beagle犬重复sc给予PEG-Det 37.5、75.0、150.0 μg/kg,随给药剂量增加,药物暴露量增大;经多次给药后,血浆中胰岛素浓度趋于平稳,体内无药物蓄积;且血糖降低幅度减少,在维持有效浓度和药效的基础上,降低了由低血糖带来的安全性风险。     

利多卡因凝胶对豚鼠的长期毒性试验研究

目的 研究30 d重复皮肤涂抹利多卡因凝胶对豚鼠产生的毒性反应。方法 利多卡因凝胶高、中、低剂量（40、20、10 g/kg）连续皮肤涂抹给药30 d,整个试验期间观察动物的临床症状,测定体质量和摄食量,分别于给药30 d及停药后14 d取部分豚鼠进行血液学、血清生化学、脏器系数和病理组织学检查。结果 豚鼠未出现因给药而引起的异常症状,各给药组豚鼠体质量增长和摄食正常;利多卡因凝胶在10、20 g/kg剂量下,豚鼠血液学、血清生化学、脏器系数和病理组织学等均未见毒理学意义的异常改变;40 g/kg利多卡因凝胶导致豚鼠血清K⁺水平,肾上腺质量及系数明显增高（P<0.05）,但脏器未出现毒性病理改变;停药14 d后,上述异常指标恢复正常,也未见其他延迟毒性。结论 在本试验条件下,皮肤涂抹利多卡因凝胶对豚鼠无明显毒性,无毒反应剂量为20 g/kg。     

酒石酸长春瑞滨胶束重复给药对Beagle犬肝、肾毒性研究

目的 研究长期重复给予注射用酒石酸长春瑞滨胶束（NVB-m）对Beagle犬肝、肾毒性的影响,同时设同类市售注射用酒石酸长春瑞滨（NVB）进行毒性比较研究。方法 Beagle犬48只,按体质量、性别随机分为对照组、空白胶束组、NVB组和NVB-m 6、12、24 mg/m²组,每组8只。NVB组给予24 mg/m²剂量的NVB,空白胶束组给予相同浓度的空白胶束,对照组给予生理盐水,静脉给药。进行一般状态观察,称取Beagle犬体质量,取血检测血清生化指标,采集尿液检测尿常规,大体解剖称量肝、肾质量,计算脏器系数并做组织病理学检查。结果 一般症状检查可见,NVB组、NVB-m 12、24 mg/m²组在给药期间有食欲不振、食量减少甚至拒食等症状,随给药次数增加而减轻。动物体质量在试验期间未见明显异常。血清生化检查可见,与对照组比较,给药期间NVB组及NVB-m 6、12、24 mg/m²组天冬氨酸转氨酶（AST）、天冬氨酸转氨酶（ALT）、总胆红素（TBIL）升高,总蛋白（TP）、白蛋白（ALB）降低,其中NVB-m 24 mg/m²组对动物TP影响较NVB组轻微;NVB组及NVB-m 24 mg/m²组动物ALT升高（部分组别部分时间差异显著P<0.05、0.01）,恢复期可恢复正常。尿生化检查、大体解剖及脏器质量检查、组织病理学检查未见明显异常。结论 NVB-m长期给药对Beagle犬有轻微肝毒性,主要表现为肝功指标异常;NVB-m对Beagle犬无肾毒性;NVB-m与NVB没有明显肝、肾毒性差异。     

醒脑静注射液对Beagle犬心血管和呼吸系统功能的影响

目的 研究醒脑静注射液对Beagle犬心血管和呼吸系统的影响。方法 已成功埋入植入子的Beagle犬6只,拉丁方设计给药,设溶媒对照组,辅料对照组,醒脑静注射液1（XNJI1,含人工麝香）高、中、低剂量（1.41、0.71、0.35 mg/kg）组和醒脑静注射液2（XNJI2,含天然麝香）高、中、低剂量（1.46、0.73、0.36 mg/kg）组,XNJI1高、中、低剂量均约为人临床等效剂量的5.2、2.6、1.3倍;XNJI2高、中、低剂量均约为人临床等效剂量的5.1、2.6、1.3倍。辅料对照组给予等体积的0.5%聚山梨酯80溶液;溶媒对照组给予等体积氯化钠注射液。前肢iv给药,每2个给药组设1 d洗脱期。利用DSI遥测系统连续测量给药前1 h至给药后4 h的心电、血压、呼吸等指标。结果 各给药组及辅料对照组部分Beagle犬出现明显过敏症状,在给药后均出现持续10~45 min的口鼻部发红肿胀、瘙痒,部分表现烦躁不安,辅料对照组最为明显。与溶媒对照组比较,XNJI1高剂量组及辅料对照组心率显著升高（P<0.05、0.01）,分别于给药后30 min、1 h恢复至给药前水平;辅料对照组给药后5、10、30 min QTc间期显著延长（P<0.01）;XNJI1高剂量组给药后10 min、0.5 h呼吸频率（BPM）显著升高,XNJI2高剂量组给药后5 min BPM显著升高（P<0.05、0.01）,辅料对照组给药后BPM明显升高但无显著差异。XNJI1比XNJI2在心率、QTc间期、BPM等方面波动较大,但二者无显著差异。结论 给予大剂量醒脑静注射液,Beagle犬出现不同程度类过敏反应症状及心电呼吸影响可能与辅料聚山梨酯80有关,临床使用应密切监测相关指标以确保用药安全。     

新型国产重组甘精胰岛素对1型糖尿病模型大鼠的降糖作用研究

目的 探讨新型国产重组甘精胰岛素注射液对1型糖尿病模型大鼠的降糖作用。方法 选择健康SD大鼠和链脲佐菌素诱导的1型糖尿病大鼠,甘精胰岛素注射液（来得时）为阳性对照药,健康SD大鼠单次sc 2、4 IU/kg剂量的重组甘精胰岛素注射液,大鼠尾静脉采血,分别测定0 h（给药前）及给药后1、2、4、6、8 h空腹血糖;1型糖尿病模型大鼠连续5 d sc 4、8 IU/kg剂量的重组甘精胰岛素注射液,每天分别测定给药前（0 h）和给药后（4 h）空腹血糖,给药第1、5天测定给药后1、2、4、6、8 h空腹血糖。结果 健康大鼠sc重组甘精胰岛素注射液2、4 IU/kg后,血糖值在2 h下降到最低,与对照组比较差异显著（P<0.01）;模型大鼠连续5 d sc 4、8 IU/kg重组甘精胰岛素注射液,血糖于给药后4~6 h下降至最低点,与模型组比较差异显著（P<0.01）;每天给药4 h后血糖均呈平稳下降趋势,与模型组比较差异显著（P<0.01）,8 IU/kg血糖值与对照组接近。结论 国产重组甘精胰岛素注射液对1型糖尿病模型大鼠有较好的降糖效果,与同剂量阳性药降糖效果相当。     

Structural characterization of insulin NPH formulations

Insulin NPH (neutral protamine hagedorn) has for long been one of the most important therapeutic formulations for the treatment of diabetes. The protracted action profile of NPH formulations is gained from crystallizing insulin with zinc in the presence of the basic poly-arginine peptide protamine. In spite of its long history and successful use, the binding mode of the insulin–protamine complex is not known. In this study, three different systems were used to study protamine binding to insulin. In the first system, crystals of an insulin–protamine complex grown in the presence of urea and diffracting to 1.5 Å resolution were analyzed. In the second system, a shorter peptide consisting of 12 arginine residues was co-crystallized with insulin in order to reduce the flexibility and thereby improve the electron density of the peptide. Both systems yielded data to a significantly higher resolution than obtained previously. In addition, a third system was analyzed where crystals of insulin and protamine were grown in the absence of urea, with conditions closely resembling the pharmaceutical formulation. Data from these NPH microcrystals could for the first time be collected to 2.2 Å resolution at a micro focused X-ray beamline. Analysis of all three crystal forms reveal potential protamine density located close to the solvent channel leading to the centrally located zinc atoms in the insulin hexamer and support that protamine binds to insulin in a not well defined conformation.     

Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats

Objective:

To evaluate the concomitant administration of methotrexate and curcumin for antiarthiritic activity in rats.

Materials and Methods:

Arthritis was induced in rats following a single subplantar injection of Freund''s complete adjuvant (0.1 ml). Rats were divided into six groups of six animals each. Group I and II were control injected with saline and Freund''s complete adjuvant (0.1 ml), respectively. Group III arthritic rats were treated with curcumin (100 mg/kg, i.p.) on alternate days. Group IV received methotrexate (MTX) (2 mg/kg, i.p.) once in a week. Group-V and VI were treated with MTX (1 mg/kg, i.p.) once in a week and after 30 min received curcumin (30 mg/kg and 100 mg/kg, thrice a week, i.p.) from 10^th to 45^th days, respectively. Body weight and the paw volume was measured on 9^th, 16^th, 23^rd, 30^th, 37^th, and 45^th days. Determination of complete blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentration was determined on the 46^th day.

Results:

An improvement in body weight and a significant (P < 0.05) reduction in arthritis was observed with the combination treatment as compared to the positive control. A significant improvement in the hematological profile was also observed in rats treated with curcumin and methotrexate.

Conclusion:

The study showed a significant anti-arthritic action and protection from hematological toxicity with the combination treatment of methotrexate and curcumin.     

苏苏小儿止咳颗粒对幼龄大鼠的长期毒性试验研究

目的 对苏苏小儿止咳颗粒进行幼龄SD大鼠长期毒性试验研究,为其提供临床前安全性评价。方法 苏苏小儿止咳颗粒高、中、低剂量（以生药计30、15、5 g/kg）连续ig给药30 d。观察指标包括一般观察、体质量、摄食量、行为学测试、雌二醇、睾酮、胫骨长度、尿液、血液学、凝血指标、血液生化、电解质、脏器质量、脏器系数及组织病理学检查等。结果 30 g/kg剂量组雄鼠给药期间出现体质量增长缓慢,恢复期可恢复正常。其余动物未见明显药物相关毒性反应,停药后未见迟缓毒性发生。结论 在本试验条件下,苏苏小儿止咳颗粒对幼龄大鼠的无毒反应剂量为15 g/kg,相当于临床拟用剂量的22倍。     

Insulin glargine versus NPH insulin in patients with type 1 diabetes

Type 1 diabetes is associated with a number of diabetes-related complications which may be minimized by maintaining good long-term metabolic control. The current guidelines recommend that glycosylated hemoglobin levels should be targeted to below 7.0% or 6.5% to reduce the incidence of diabetic complications, including micro- and macrovascular disorders. However, this intensive metabolic control is hindered by the occurrence of hypoglycemia. The episodes of hypoglycemia are problematic for patients taking intermediate-acting insulin preparations (i.e., NPH insulin), which have traditionally formed the mainstay basal insulin treatment. With NPH insulin, the pharmacokinetic profile is such that peak insulin activity occurs 4-6 hours following administration; therefore, nocturnal hypoglycemia commonly takes place after bedtime administration of the insulin. The development of the long-acting human insulin analogue insulin glargine now provides patients with an insulin that offers a longer duration of action (up to 24 hours) and a smoother time-action profile compared with those of NPH insulin. A number of clinical trials comparing the safety and efficacy of insulin glargine and NPH insulin in patients with type 1 diabetes show that, in addition to other clinical benefits over NPH insulin, insulin glargine may also improve glycemic control and satisfaction in this patient population.     

Increased Amyloid-β Peptide-Induced Memory Deficits in Phospholipid Transfer Protein (PLTP) Gene Knockout Mice

Oxidative stress is recognized as one of the earliest and most intense pathological processes in Alzheimer''s disease (AD), and the antioxidant vitamin E has been shown to efficiently prevent amyloid plaque formation and neurodegeneration. Plasma phospholipid transfer protein (PLTP) has a major role in vitamin E transfers in vivo, and PLTP deficiency in mice is associated with reduced brain vitamin E levels. To determine the impact of PLTP on amyloid pathology in vivo, we analyzed the vulnerability of PLTP-deficient (PLTP-KO) mice to the toxic effects induced by intracerebroventricular injection of oligomeric amyloid-β_25–35 (Aβ_25–35) peptide, a non-transgenic model of AD. Under basal conditions, PLTP-KO mice showed increased cerebral oxidative stress, increased brain Aβ_1–42 levels, and a lower expression of the synaptic function marker synaptophysin, as compared with wild-type mice. This PLTP-KO phenotype was associated with increased memory impairment 1 week after Aβ_25–35 peptide injection. Restoration of brain vitamin E levels in PLTP-KO mice through a chronic dietary supplementation prevented Aβ_25–35-induced memory deficits and reduced cerebral oxidative stress and toxicity. We conclude that PLTP, through its ability to deliver vitamin E to the brain, constitutes an endogenous neuroprotective agent. Increasing PLTP activity may offer a new way to develop neuroprotective therapies.     

Acute and sub-chronic toxicity studies of the extract of Thunberg Fritillary Bulb

The aim of this study was to investigate the acute and sub-chronic toxicity of extract of Thunberg Fritillary Bulb. For the acute toxicity tests, graded doses of the extract were administered orally to mice. The animals were observed for toxic symptoms and mortality daily for 14 days. In the sub-chronic toxicity study, rats were orally administered the extract at doses of 1 and 3 mg/kg body weight (BW) for 26 weeks. After 26 weeks, the rats were sacrificed for hematological, biochemical and histological examination. In the acute toxicity tests, the estimated median lethal dosage (LD₅₀) was 52.2 mg/kg body weight in the mice. In the sub-chronic toxicity tests, a dose of 1 mg/kg body weight presented no toxicity. Above the 1 mg/kg dose, the main adverse signs observed in male rats were body or head tremor and spontaneous motor activity reduction. There were no other significant changes observed in hematology, blood biochemistry, organ weight and organ histology. The overall findings of this study indicate that the extract of Thunberg Fritillary Bulb is non-toxic up to 1 mg/kg body weight, which can be considered a safe application dose.     

Endocrine effects of contaminated sediments on the freshwater snail Potamopyrgus antipodarum in vivo and in the cell bioassays in vitro

Lake Pilnok located in the black coal-mining region Ostrava-Karvina, Czech Republic, contains sediments highly contaminated with powdered waste coal. Moreover, population of the endangered species of narrow-clawed crayfish Pontastacus leptodactylus with high proportion of intersex individuals (18%) was observed at this site. These findings motivated our work that aimed to evaluate contamination, endocrine disruptive potency using in vitro assays and in vivo effects of contaminated sediments on reproduction of sediment-dwelling invertebrates. Chemical analyses revealed low concentrations of persistent chlorinated compounds and heavy metals but concentrations of polycyclic aromatic hydrocarbons (PAH) were high (sum of 16 PAHs 10mug/gdw). Organic extracts from sediments caused significant in vitro AhR-mediated activity in the bioassay with H4IIE-luc cells, estrogenicity in MVLN cells and anti-androgenicity in recombinant yeast assay, and these effects could be attributed to non-persistent compounds derived from the waste coal. We have also observed significant in vivo effects of the sediments in laboratory experiments with the Prosobranchian euryhaline mud snail Potamopyrgus antipodarum. Sediments from Lake Pilnok as well as organic extracts of the sediments (externally added to the control sediment) significantly affected fecundity during 8 weeks of exposure. The effects were stimulations of fecundity at lower concentrations at the beginning of the experiment followed by inhibitions of fecundity and general toxicity. Our study indicates presence of chemicals that affected endocrine balance in invertebrates, and emphasizes the need for integrated approaches combining in vitro and in vivo bioassays with identification of chemicals to elucidate ecotoxicogical impacts of contaminated sediment samples.