The Tubifex tubifex assay for the determination of acute toxicity

An express (3-minute) test for acute toxicity determination by using the oligochaete annelid, Tubifex tubifex, is described. The EC50(Tubifex tubifex) [EC50(Tt)] for movement inhibition was calculated by using a concentration-response dependence. The reproducibility of the test was checked over several years and by several workers. Its applicability is limited to compounds which are soluble in water. The calculated EC50(Tt) indices correlate with LC50 values determined by using the fish, Pimephales promelas (96-hour assay), and with ICG50 values determined by using the ciliate, Tetrahymena pyriformis (48-hour assay) with high statistical significance (r = 0.822, n = 35, and r = 0.927, n = 80, respectively). The correlation between the EC50(Tt) indices and rat oral LD50 values (48-hour assay) was r = 0.519 (n = 67). The correlation within organic compounds was closer (r = 0.635, n = 60) than with the heterogeneous series of chemicals. A similar trend was noticed for the correlation with mouse oral LD50 values (r = 0.479, n = 56) with the heterogeneous series of chemicals, as compared that with the series without inorganic salts (r = 0.605, n = 42), and similarly with mouse intraperitoneal LD50 values, where r = 0.543 (n = 50) with the heterogeneous series of chemicals and r = 0.893 (n = 33) with the series of organic chemicals.     

Lack of predictivity of the rat lethality (LD50) test for ecological and human health effects

The relationship between acute toxicity in rats (LD50 values) and indicators of potential health hazards in humans was investigated, based on a chemical population-based paradigm (i.e. the "chemical diversity approach"). These structure-activity relationship-based analyses indicate that high toxicity in rats (i.e. a low LD50 value) is not a good predictor of health effects in humans. In fact, it was found that high acute toxicity to minnows, as well as toxicity to cultured cells, showed significantly greater associations with the potential for health effects than rat LD50 values.     

Prediction of acute toxicity in HPCT-1E3 hepatocytoma cells with liver-like transport activities

A battery of in vitro methods has been developed for the prediction of acute oral toxicity, to reduce the number of animals used for this purpose. However, the results of these tests correlate more closely with lethal serum concentrations than with lethal doses. To address this issue, we have further evaluated the HPCT-1E3 model, which may be better able to emulate toxicokinetic factors that occur in vivo, due to the presence in these hepatocytoma cells of endogenous transmembrane carriers and a basal activity of xenobiotic metabolism. IC50 values produced by using the MTT test after a 48-hour incubation with 20 randomly-selected MEIC substances, correlated better with human oral LD50 values than with LC50 data, supporting this hypothesis. As with other models, the toxicity of receptor-specific rather than cytotoxic substances, for example digoxin, was underpredicted. When digoxin was removed from the correlation analysis, the coefficient of determination (r(2)) improved to 0.81, and none of remaining chemicals were wrongly predicted by more than one order of magnitude. IC50 values obtained with HepG2 cells under similar conditions (MEIC Test No. 3, 24 hours, MTT) correlated with human LD50 data with a r(2) value of 0.55. A direct comparison of HPCT-1E3 and HepG2 cells further suggested that the differences between them may be due to transport processes. In conclusion, the HPCT-1E3 model may be valuable in improving the prediction of lethal doses, rather than lethal serum concentrations.     

Daily rhythms of toxicity and effectiveness of anesthetics (MS222 and eugenol) in zebrafish (Danio rerio)

Although the chronotoxicity of xenobiotics is relatively well known in mammals, the existence of daily rhythms of drug toxicity and effectiveness in fish has been neglected to date. The aim of this research was to investigate the influence of the time (middle of the light phase [ML] versus middle of the dark phase [MD]) of exposure to two anesthetic substances (MS-222 or clove oil) commonly used with fish on the median lethal concentration (LC(50)) and swimming activity of zebrafish (Danio rerio). To this end, adult zebrafish were kept under a 12 h:12 h light-dark (LD) cycle and exposed to different concentrations of the anesthetics for 15 min at ML or MD. LC(50) calculations were performed using the Spearman-Karber program, whereas swimming activity was video-recorded and analyzed with specialized software. Zebrafish exhibited a mostly diurnal activity pattern (77.9% of activity occurring during daytime). The acute toxicity and mortality caused by MS-222 and eugenol varied with the time of exposure. For MS-222, the LC(50) was 170.6 ± 7.4 mg/L in fish exposed at ML and 215.6 ± 3.9 mg/L at MD, whereas for eugenol the LC(50) was 70.3 ± 3.1 mg/L at ML and 104.9 ± 5.4 mg/L at MD. Exposure to sublethal concentrations of MS-222 and eugenol altered the swimming patterns of zebrafish in a different manner depending on the time of exposure. Thus, the time required for decreasing swimming activity during exposure to anesthetics was shorter at ML than at MD, whereas the recovery period was longer during the day. In conclusion, these results revealed that the toxicity and effectiveness of both anesthetic substances is highest during daytime, the active phase of fish, thus suggesting a link between the daily rhythms of behavior and toxicity.     

Reversible AChE inhibitors in C. elegans vs. rats, mice

We are investigating whether Caenorhabditis elegans could be used as a screen for vertebrates by comparing the responses of components of its cholinergic system to well-characterized toxicants. We assessed whether C. elegans displays similar toxicity as rats and mice to reversible acetylcholinesterase (AChE) inhibitors, and sought to corroborate that the toxicity mechanism is the same. To determine relative potencies, movement-concentration curves were generated, 50th percentiles for movement were located, ranked and compared statistically to rat and mouse oral acute LD50s. The ranking was significantly correlated to rat and mouse rankings (alpha=0.05). We measured a concentration-dependent decrease in AChE activity correlating to a decrease in movement for each carbamate, suggesting that the mechanism of toxicity is the same. Finally, as seen in mammals, inhibition of AChE activity occurred before a movement decrease. The response of C. elegans to carbamate exposure shows significant correlation to rat and mouse data.     

噻吩磺隆的毒性及致突变性

选用大鼠、豚鼠及家兔,采用经口及皮肤,粘膜染毒途径,研究其急性毒性。同时有Ames试验,小鼠骨髓嗜多染红细胞微核试验及小鼠睾丸初级精母细胞染色体畸变试验进行致突变性研究,了解噻吩磺隆的毒性及致突变性。大鼠急性经口LD50大于5000mg／kg,经皮LD50大于2000mg／kg。家兔皮肤刺激试验阴性,轻度眼刺激性和弱致敏性。Ames试验,微核试验及小鼠睾丸初级精母细胞染色体畸变试验结果均为阴性。结论 噻吩磺隆属低毒性农药,在本实验条件下无致突变作用。     

Acute and Cumulative Effects of Unmodified 50-nm Nano-ZnO on Mice

Nanometer zinc oxide (nano-ZnO) is widely used in diverse industrial and agricultural fields. Due to the extensive contact humans have with these particles, it is crucial to understand the potential effects that nano-ZnO have on human health. Currently, information related to the toxicity and mechanisms of nano-ZnO is limited. The aim of the present study was to investigate acute and cumulative toxic effects of 50-nm unmodified ZnO in mice. This investigation will seek to establish median lethal dose (LD50), a cumulative coefficient, and target organs. The acute and cumulative toxicity was investigated by Karber’s method and via a dose-increasing method, respectively. During the experiment, clinical signs, mortality, body weights, hematology, serum biochemistry, gross pathology, organ weight, and histopathology were examined. The LD50 was 5177-mg/kg·bw; the 95% confidence limits for the LD50 were 5116–5238-mg/kg·bw. It could be concluded that the liver, kidney, lung, and gastrointestinal tract were target organs for the 50-nm nano-ZnO acute oral treatment. The cumulative coefficient (K) was 1.9 which indicated that the cumulative toxicity was apparent. The results also indicated that the liver, kidney, lung, and pancrea were target organs for 50-nm nano-ZnO cumulative oral exposure and might be target organs for subchronic and chronic toxicity of oral administered 50-nm ZnO.     

使它隆对草鱼、鲢鱼和鲫鱼的急性毒性

【背景】有关农药对水生生物的毒性和影响已有大量的报道,但关于使它隆对水生生物影响的研究较少。【方法】采用半静态法测试了使它隆对草鱼、鲢鱼和鲫鱼的急性毒性,并计算使它隆对草鱼、鲢鱼和鲫鱼的安全浓度。试验过程中,水温保持在25℃±1℃。【结果】随着受试鱼苗在药液中暴露时间的延长,LC50值逐渐减小。使它隆对草鱼、鲢鱼和鲫鱼的96h LC50值分别为0．4764、0．3962和0．6918mg·L^-1。使它隆对草鱼、鲢鱼和鲫鱼的安全浓度分别为0．0476、0．0392和0．0692mg·L^-1【结论与意义】使它隆对草鱼、鲢鱼和鲫鱼表现高毒,这为使它隆的风险评估和环境安全管理提供了依据。     

Alternative methods for the median lethal dose (LD(50)) test: the up-and-down procedure for acute oral toxicity

The authors have developed an improved version of the up-and-down procedure (UDP) as one of the replacements for the traditional acute oral toxicity test formerly used by the Organisation for Economic Co-operation and Development member nations to characterize industrial chemicals, pesticides, and their mixtures. This method improves the performance of acute testing for applications that use the median lethal dose (classic LD50) test while achieving significant reductions in animal use. It uses sequential dosing, together with sophisticated computer-assisted computational methods during the execution and calculation phases of the test. Staircase design, a form of sequential test design, can be applied to acute toxicity testing with its binary experimental endpoints (yes/no outcomes). The improved UDP provides a point estimate of the LD50 and approximate confidence intervals in addition to observed toxic signs for the substance tested. It does not provide information about the dose-response curve. Computer simulation was used to test performance of the UDP without the need for additional laboratory validation.     

Anticonvulsant and toxicity evaluation of some 7-alkoxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline-1(2H)-ones

To further investigate anticonvulsant activity of quinoline derivatives, a series of 7-alkoxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline-1(2H)-one derivatives was synthesized starting from 7-hydroxyl-3,4-dihydro-2(1H)-quinoline. In initial (phase I) screening and quantitative (phase II) evaluation, compound 7-benzyloxyl-4,5-dihydro-[1,2,4]thiazolo[4,3-a]quinoline-1(2H)-one (3f) was among the most active but also has the lowest toxicity. In the anti-MES potency test, it showed median effective dose (ED(50)) of 12.3 mg/kg, median toxicity dose (TD(50)) of 547.5 mg/kg, and the protective index (PI) of 44.5, which is much greater than PI of the prototype drugs phenytoin, phenobarbital, carbamazepin, and valproate. Compound 3f was chosen for further evaluation. In phase III pharmacological test, the compound had median hypnotic dose (HD(50)) and median lethal dose (LD(50)) of 1204 mg/kg and >3000 mg/kg, respectively, thus demonstrating much greater margin of safety compared to prototype drugs. The compound 3f also showed significant oral activity against MES-induced seizures and low oral neurotoxicity in mice in phase IV pharmacological test. Possible structure-activity relationship was discussed.     

Acute toxicity of bisphenol A in rats

Bisphenol A (BPA), an estrogenic compound, is used in manufacturing plastics and is known to produce toxic effects on various systems in man and animals. Since the use of plastics in day-to-day life is increasing, exposure to BPA will also increase. Therefore, this study was undertaken to determine the median lethal dose (LD50) of BPA via intraperitoneal and intravenous route in adult rats (by Dixon's up and down method) and also to know the acute systemic changes (in blood pressure, respiration and ECG) produced by lethal dose of BPA. Adult female albino rats of Charles Foster strain were used in the study. LD50 of BPA was 841 and 35.26 mg/kg body weight for ip and iv route, respectively. Injection of lethal dose of BPA (40 mg/kg body weight) produced acute toxicity manifesting as immediate respiratory arrest and hypotension after the injection of BPA followed by bradycardia. The animals died within 7.3 +/- 0.7 min. Volume of ethanol (vehicle; 0.1 mL) present in the lethal dose of BPA was not lethal and had no effect on respiration, blood pressure and heart rate. The results provide evidence that the acute exposure to BPA produces lethality with a very narrow range of lethal and survival dose for iv route. Further, the lethality appears to be due to respiratory arrest and hypotension.     

卵孢白僵菌及添加杀虫助剂对两种地老虎的生物活性测定

【目的】确定卵孢白僵菌Beauveria brongniartii菌株NEAU30503对八字地老虎Xestia c-nigrum(Linnaeus)和小地老虎Agrotis ypsilon(Rottemberg)的杀虫活性。【方法】采用浸叶法测定NEAU30503对地老虎低龄幼虫的生物活性,土壤处理法测定对高龄幼虫的杀虫活性。【结果】NEAU30503对5日龄八字地老虎幼虫第15天毒力回归方程为y=0.6568x-0.1636(r=0.9846),其LC_(50)、LC_(80)分别为7.28×10~7孢子/m L、139.14×10~7孢子/m L;对5龄八字地老虎幼虫毒力回归方程为y=1.0929x-3.2893(r=0.9801),其LC_(50)、LC_(80)分别为3.85×10~7孢子/L、22.65×10~7孢子/L。未死幼虫化蛹后仍有部分蛹死于白僵菌感染,并能明显降低成虫羽化率。白僵菌与Bt、茶皂素、以及亚致死剂量的高效氯氢菊酯和阿维菌素混用能明显提高其杀虫活性和杀虫速度。【结论】NEAU30503对地老虎具有较高的杀虫活性,土壤处理防治高龄幼虫效果好,喷雾处理防治低龄幼虫加入少量的高效氯氢菊酯或茶皂素效果明显。本研究为开展白僵菌田间防治地老虎提供了科学依据。     

5%甲氨基阿维菌素苯甲酸盐微乳剂对6种环境生物的急性毒性

甲氨基阿维菌素苯甲酸盐是一种新型的抗生素类杀虫剂、杀螨剂,其大量使用可能会导致一系列的生态风险,因此有必要开展其对相关环境生物毒性的研究。测定了甲氨基阿维菌素苯甲酸盐对意大利蜜蜂、日本鹌鹑、斑马鱼、家蚕、大型溞和赤子爱胜蚓6种非靶标生物的急性毒性。5％甲氨基阿维菌素苯甲酸盐微乳剂对蜜蜂的急性经口LC50（48 h）为0．555 a．i．mg· L-1,对鹌鹑的经口LD50（7 d）为148．369 a．i．mg· kg-1,对斑马鱼的LC50（96 h）为0．368 a．i．mg· L-1,对家蚕的急性摄入毒性LC50（96 h）为0．005 a．i．mg· L-1,对大型溞的运动抑制毒性EC50（48 h）为0．020 a．i． mg· L-1,对蚯蚓的急性毒性LC50（14 d）为18．397 a．i．mg· kg-1。该农药对家蚕和大型溞均为剧毒,对蜜蜂和斑马鱼均为高毒,对鹌鹑中毒,对蚯蚓低毒。总体而言,甲氨基阿维菌素苯甲酸盐微乳剂对环境生物危害大,在使用过程中要注意。     

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

The Registry of Cytotoxicity: toxicity testing in cell cultures to predict acute toxicity (LD50) and to reduce testing in animals

This is a translation of a report on the Registry of Cytotoxicity (RC), originally published in German in 1998. The report presented an advanced in vitro method, which can significantly reduce the number of animals needed for the toxicity testing of a broad range of compounds/xenobiotics. With the RC method, it was possible to predict the oral or intravenous acute toxicity (LD50)--which is a regulatory requirement for newly developed pharmaceuticals and industrial and household chemicals--from the cytotoxicity data (mean IC50 = IC50X) obtained with mammalian cells. The RC method can be used before the in vivo test, and it does not pose any additional harm or suffering to laboratory animals. The RC method is of broad practical use: it can be applied, for example, in the pharmaceutical industry or the chemical industry in regulatory testing or in research. It is ready for validation, and could then be incorporated into OECD guidelines, thus reducing the total number of animals needed for regulatory toxicity testing. The RC method is based on the comparison of the IC50X values and the LD50 values by using linear regression analysis. With the RC method, it was possible to predict, within a predefined dose range, the acute oral LD50 for 252 of 347 xenobiotics, and the intravenous LD50 for rats and/or mice for 117 of 150 xenobiotics. Comparative studies showed that these results are highly reproducible.     

Lethal synergism of phencyclidine with a precursor and contaminant, 1-piperidinocyclohexanecarbonitrile

The acute median lethal dose (LD50) was determined in rats (p.o.) and mice (i.p.) for phencyclidine (PCP) or 1-piperidinocyclohexanecarbonitrile (PCC) individually and in combination. The LD50 (mg/kg) of PCP was 2.3 or 2.5 times higher than the LD50 of PCC in mice or rats, respectively. For mice, combinations (8:1 or 4:1) of PCP + PCC showed additive synergism, while in rats a 2:1 combination caused sub-additive synergism. Dogs receiving an intravenous infusion of PCP + PCC (4:1) died at a one-third lower dosage of PCP than when PCP was given alone. Prior treatment with NaNO₂ significantly elevated the i.p. LD50's in mice for PCC alone or PCP + PCC (3:1) as it did for KCN. These data support the inference that toxicity from PCC arises primarily from the cyanide ion which it releases. The possibility of a toxic synergism in the course of human exposure to PCC-contaminated “street PCP” is raised.     

2，4-D二甲胺盐对草鱼、鲢鱼和鲫鱼的急性毒性

【背景】2,4-D二甲胺盐是一种选择内吸性除草剂,用于清除果园、牧场、水域等环境中的藜、苋等阔叶杂草。本文明确了2,4-D二甲胺盐在防除鱼塘杂草过程中对淡水鱼类的毒性。【方法】采用半静态法测试了2,4-D二甲胺盐对草鱼、鲢鱼和鲫鱼的急性毒性,并明确了2,4-D二甲胺盐对草鱼、鲢鱼和鲫鱼的安全浓度。【结果】随着供试鱼苗在药液中暴露时间的延长,LC50。值逐渐减小。2,4-D二甲胺盐对草鱼、鲢鱼和鲫鱼的96hLC50值分别为369．27、339．20和438．47mg·L^-1。2,4-D二甲胺盐对草鱼、鲢鱼和鲫鱼的安全浓度分别为36．93、33．92和43．85mg·L^-1。【结论与意义】2,4-D二甲胺盐对草鱼、鲢鱼和鲫鱼表现低毒。本研究为合理使用2,4一D二甲胺盐防除水生杂草提供了依据。     

Insecticidal activity of selected monoterpenoids and rosemary oil to Agriotes obscurus (Coleoptera: Elateridae)

Acute toxicities of three naturally occurring monoterpenoid essential oil constituents and the essential oil of rosemary were tested against late instars of Agriotes obscurus (L.) (Coleoptera: Elateridae). Both contact and volatile toxicities of thymol, citronellal, eugenol, and rosemary oil were determined. Also, phytotoxicity of these compounds was evaluated on corn germination and seedling development. Thymol had the greatest contact toxicity (LD50 = 196.0 microg/larva), whereas citronellal and eugenol were less toxic (LD50 = 404.9 and 516.5 microg/larva, respectively). Rosemary oil did not show any significant contact toxicity, even at 1,600 microg/larva. In terms of volatile toxicity, citronellal was the most toxic to wireworm larvae (LC50 = 6.3 microg/cm3) followed by rosemary oil (LC50 = 15.9 microg/cm3), thymol (LC50 = 17.1 microg/cm3), and eugenol (LC50 = 20.9 microg/cm3). Thymol, eugenol, and citronellal significantly inhibited corn seed germination and development, whereas rosemary oil had only minimal phytotoxic effects.     

Effects of alpha-mangostin from mangosteen pericarp extract and imidacloprid on Nilaparvata lugens (Stal.) and non-target organisms: toxicity and detoxification mechanism

The brown planthopper, Nilaparvato lugens Stat. (BPH) is the most devastating insect pest in rice fields. Outbreaks of BPH, which are resistant to many synthetic insecticides, can cause total rice crop loss. This research was done to evaluate the efficiency of extracts of mangosteen pericarp (Garcina mangostana L.) as an alternative control of BPH Thailand strain. Topical spraying was applied to various stages of nymphal and adult BPH to determine toxicity. An ethanol extract of mangosteen pericarp extract gave the best control of BPH, with LC50 of 4.5% w/v (r2 = 0.95) with 3rd instar BPH nymphs when compared with the other solvents, hexane, acetone and dichloromethane. The active compound, alpha-mangostin showed an LC50 of 5.44%w/v (r2 = 0.88). The toxicity of this extract was less than that of Imidacloprid which showed an LC50 of 0.0042% w/v (r2 = 0.99). The toxicity to non-target organisms was determined. This extract showed toxicity to guppies ((LC50 = 2.53 and 4.27 ppm for females and males, respectively; r2 = 0.97 and 0.97, respectively), bees (LC50 = 4.38% w/v, r2 = 0.95) and mice (no oral acute toxicity and no dermal inflammation but showed eye irritation in 1 day which became normal within 3 days). In vitro detoxification enzyme activities of carboxylesterase, acetylcholinesterase and glutathione-s-transferase from BPH after 24 hours exposure were also observed. Carboxylesterase showed stronger activity than other enzymes. Toxicity in terms of LC50 values of both the extract and imidacloprid treatments increased in each generation. The LC50 values for each generation were 4.22-6.67 after sequential spraying. After the ethanol extract was kept at 4 degrees C, room temperature and 55 degrees C for 3 months, the quantity of alpha-mangostin and the BPH control efficiency was lower at 55 degrees C than those for other temperatures. The results from this research indicate that mangosteen pericarp extract can be an alternative insecticide for the control of BPH, which may possess high efficiency, cause with fewer environmental problems and result in less resistance in the BPH.