A statistical evaluation of the fixed dose procedure

The Fixed Dose Procedure (FDP) was first proposed in 1984 by the British Toxicology Society, as an alternative to the conventional LD50 test (OECD Test Guideline 401), for determining acute oral toxicity. The FDP used fewer animals and caused less suffering than the LD50 test, and provided information on acute toxicity which allowed substances to be classified according to the European Union hazard classification system. In 1992, the FDP was introduced as OECD Test Guideline 420. In 1999, as part of an initiative to phase out Test Guideline 401, a review of the FDP was undertaken. The aim of the review was to provide further reductions and refinements, and classification according to the criteria of the Globally Harmonised Hazard Classification and Labelling Scheme. The revised FDP was adopted by the OECD in 2001. This article concerns the development and revision of the FDP. It illustrates how statistical modelling and simulation can be used to increase the efficiency of a test procedure and reduce the number of animals needed for an in vivo validation of the procedure.     

Refinement and reduction of acute oral toxicity testing: a critical review of the use of cytotoxicity data

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300 mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.     

上下法在急性毒性试验中的应用进展

随着全球对动物福利的关注,各种传统急性毒性试验的替代方法得到了人们越来越广泛的关注。上下法作为OECD修订发布的急性毒性试验方法之一,是传统急性毒性试验的一个替代方法,有着巨大的应用价值和发展前景。本文简要介绍了该方法的发展历程和在实际应用中的注意事项,评价了上下法与传统方法及其他替代方法相比的优缺点,并综述了近年来该方法在国内外的应用进展。     

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.     

Toxicity of γ-hexachlorocyclohexane (Lindane) to Gammarus pulex

SUMMARY. The acute toxicity of the pesticide γ-hexachlorocyclohexane (Lindane) to Gammarus pulex was determined by a standard procedure and by a modification of the conventional toxicity test protocol. Animals were exposed to a range of concentrations for various periods of time from 1 to 1000 min and transferred to clean water. Animals continued to die for up to 3 weeks after the initial exposure to the poison. Toxicity curves were obtained expressing the relationships between concentration of poison and median survival time (LT50) and between concentration of poison and median lethal exposure time, i.e., the duration of exposure required to cause the eventual death of half the animals. For a given concentration of pesticide, the duration of exposure which will cause 50% mortality is far less than is indicated by a conventional toxicity test in which animals are continuously exposed to the poison. It is suggested that the results of conventional acute lethal toxicity tests may have limited predictive value even when applied to field situations involving the discharge of lethal levels of pollutants to receiving waters.     

乙腈的水生态基准

本文参照美国国家环保局推荐的“推导保护水生生物及其用途的国家水质基准的技术指南”,根据我国水生生物区系特点,通过水生生物毒性试验研究和制定石油化工废水中重要污染物—乙腈的水生态基准。试验动物涉及到4个门、6个纲、8个科、13属和13个种。文中根据乙腈对13种水生动物的急性毒性试验,对水生动物的慢性毒性试验以及对水生植物浮萍的生长抑制试验,推导出乙腈的基准连续浓度为413mg／L,基准最大浓度为1145mg／L。     

Is essential oil production influenced by air pollution in Ocimum basilicum L.?

Air pollution elucidates a wide range of symptoms in higher plants. Relatively little information is available, however, on how airborne toxic chemicals influence the production of bioactive compounds in medicinal plants. In our study, the No. 227 OECD Guideline for the Testing of Chemicals: Terrestrial Plant Test: Vegetative Vigour Test was followed to assess the deleterious effects of a diesel exhaust emission aerosol sample under laboratory conditions. Ocimum basilicum L. was used as test plant. Level of two bioactive compounds, eugenol and linalool was determined both in the control and in the treated samples using HPLC. While eugenol content remained unchanged, linalool content showed a significant increase after the treatment.     

Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT)

Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.     

Effects of environmentally hazardous chemicals on the emergence and early growth of selected Australian plants

The effect of soil-incorporated copper, tri-allate, and anthracene on the emergence and early growth of three Australian native species (Banksia ericifolia, Casuarina distyla andEucalyptus eximia) and three crop species (Avena sativa, Cucumis sativus andGlycine max), was assessed using OECD Test Guideline 208. The crop species are sensitive species used in overseas phytotoxicity testing, and their responses were compared with those of the native species. Seeds were grown in pots in a glasshouse in a sandy loam soil at the chemical concentrations of 0, 10, 100, 1000 and 2000 mg kg^–1. LC50 and EC50 values were determined for each species. The most sensitive species was the monocotyledonA. sativa, while among the five dicotyledonsC. distyla was most sensitive. All three chemicals delayed emergence and affected seedling growth. The results indicate that the conditions of the OECD Test Guideline can be met under Australian conditions, but that the Guideline requires modification for use with Australian native species.     

Critique on the Use of the Standardized Avian Acute Oral Toxicity Test for First Generation Anticoagulant Rodenticides

Avian risk assessments for rodenticides are often driven by the results of standardized acute oral toxicity tests without regards to a toxicant's mode of action and time course of adverse effects. First generation anticoagulant rodenticides (FGARs) generally require multiple feedings over several days to achieve a threshold concentration in tissue and cause adverse effects. This exposure regimen is much different than that used in the standardized acute oral toxicity test methodology. Median lethal dose values derived from standardized acute oral toxicity tests underestimate the environmental hazard and risk of FGARs. Caution is warranted when FGAR toxicity, physiological effects, and pharmacokinetics derived from standardized acute oral toxicity testing are used for forensic confirmation of the cause of death in avian mortality incidents and when characterizing FGARs’ risks to free-ranging birds.     

菲、芘、1,2,4-三氯苯对土壤高等植物根伸长抑制的生态毒性效应

测定了草甸棕壤条件下,菲、芘、１,２,４—三氮苯对高等植物（小麦、白菜、西红柿）根伸长抑制串以及复合污染毒性效应。结果表明,菲、芘、１,２,４—三氮苯浓度与植物根伸长抑制串呈显著线性或对数相关（ｐ＝０．０５）。３种化学品对植物根伸长抑制的强弱顺序为１,２,４—三氮苯＞菲＞芘。这与３种化学品的水中溶解度大小显著相关。小麦是３种供试植物中对有机污染物最敏感植物。菲、芘、１,２,４—三氮苯复合污染主表现为协同作用。     

The equivalence of assays within individual guidelines for the testing of the potential mutagenicity of chemicals: problems associated with battery selection

Many individual Mutagenicity Guidelines contain suggested test systems with choices of such parameters as strains, cell types and even endpoint assayed. Comparisons have been made of data obtained from variants of yeast assays for the induction of mitotic recombination, in vitro assays for the induction of chromosome aberrations and assays for the induction of cell transformation. Individual test variants included in guidelines of the EEC and OECD show considerable qualitative and quantitative variability of response to potential mutagens and carcinogens. Such variability between assays within the same guideline raises considerable problems in the selection of test batteries chosen from published Mutagenicity Guidelines. Improved battery selection is dependent upon the reduction of choice within guidelines to those assays which produce consistent and reproducible results.     

Biometric Evaluation of the ATC Method for the Determination of the Acute Dermal Toxicity of Chemicals

The new dermal acute toxic class (ATC) method is presented for one specific classification system for chemicals (released by the European Union) according to acute dermal toxicity. It is a stepwise procedure using three animals of one sex per step. Three starting doses are possible. Assuming a Probit model for the dose response relationship probabilities of a correct, of a less and of a more stringent classification are calculated. It is shown that these probabilities depend only weakly on the starting dose. Also, the expected numbers of used and of dead animals are derived in dependence on the LD₅₀ and on the dose response slope β, as well as the starting doses minimizing the expected animal number are proposed. The results demonstrate that the dermal ATC method is a reliable alternative to the classical LD₅₀ test with the use of significantly fewer animals.     

复合生物杀菌剂F6-11的安全性毒理学研究

目的：为研究复合生物杀菌剂F6．11毒性,采用动物实验法进行毒理学评价。方法：对复合生物杀菌剂F6-11进行小鼠急性毒性试验、小鼠骨髓嗜多染红细胞微核试验、亚急性毒性试验、家兔多次完整皮肤刺激试验、急性眼刺激试验、豚鼠皮肤变态反应试验、鱼类延长毒性14天试验。结果：复合生物杀菌剂F6—11对小鼠急性毒性LD50〉5000mg／kg．bw,属于实际无毒级物质;小鼠微核试验该杀菌剂各剂量组与阴性对照组比较,微核率无显著性差异（P〉0．05）;亚急性毒性试验动物血常规、生化指标及各脏器均未发现异常;对家兔多次完整皮肤及眼刺激反应积分均为0,均属无刺激性;对豚鼠皮肤变态反应试验组动物与阴性对照组无可见不同．试验组动物皮肤致敏反应积分为0,无致敏作用;鱼类延长毒性14天试验无异常。结论：毒理学研究表明,复合生物杀菌剂F6．11具有良好的使用安全性。     

PFOS对蚯蚓急性毒性和回避行为的影响

全氟辛烷磺酰基化合物(PFOS)作为一种新型持久性有机污染物,已经成为环境科学和毒理学的研究热点,其对生态环境的影响值得深入研究.本文采用OECD标准滤纸接触法、人工土壤法及自然土壤法研究了PFOS对蚯蚓急性致死作用及回避行为的影响.结果表明： PFOS对蚯蚓的急性毒性作用与染毒时间和染毒浓度相关,试验求得滤纸法48 h、人工土壤法14 d和自然土壤法14 d的LC₅₀值分别为13.64 μg·cm^-2、955.28 mg·kg^-1和542.08 mg·kg^-1;人工土壤和自然土壤中蚯蚓在PFOS的最大试验浓度组160 mg·kg^-1中均表现出显著的回避行为,表明蚯蚓可以明显感知较高浓度PFOS污染土壤并作出回避反应.与急性毒性试验的测试终点LC₅₀相比,蚯蚓行为测试终点对PFOS的反应更为敏感.自然土壤中PFOS对蚯蚓的急性毒性大于人工土壤,相同浓度PFOS作用下,蚯蚓于自然土壤中的回避行为较人工土壤明显.     

斑马鱼胚胎发育技术在毒性评价中的应用

对斑马鱼（Brachydanio rerio)胚胎发育技术在环境科学领域的应用作一综述,斑马鱼胚胎毒性技术是各国际标准组织认可的标准毒性测定方法之一,属于致畸效应检验,该项技术成本低、易操作、灵敏度高,特别是具有可记录多项毒性指标的特点,并可以此判断污染物的致毒机理,斑马鱼胚胎发育过程受重金属影响较大,其中Cu的毒性最强,Hg次之,Cr最弱,有机农药中三苯基锡类（TPTA）的毒性最强,林丹次之;有机试剂中含卤素取代基和苯胺类毒性最大,这与其它毒性测定方法的结果完全一致,并表现出较高的灵敏度,特别是选用非致死性的指标,可以初步认定这对测定复合污染物毒性并分析毒物的致畸效应方面有很好的发展前景。     

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.     

A protocol for conducting 7-day daily renewal tests with Lemna gibba

Lemna gibba (a duckweed) is a freshwater macrophyte commonly used in toxicity testing, and Lemna spp are currently the only aquatic higher plants required for evaluation of pesticides under the pesticide registration guidelines of the EPA. The methods currently available for toxicity testing by various organizations and agencies, including ASTM, OECD, EPA and Environment Canada, are largely static or semistatic tests with unspecified renewal intervals (OECD) and may not provide a consistent means of exposure owing to short toxicant half-life in aquatic media, uptake of chemical by plants and evaporation of nutrient media. The procedure outlined here details a simple and efficient 7-day daily static renewal procedure for conducting toxicity tests with L. gibba, the appropriate end points to assess, the statistical criteria necessary for analyzing the toxicity data, as well as the steps required to culture and maintain L. gibba. This protocol is based on a modified version of a widely accepted static method.     

The bovine corneal opacity and permeability test in routine ocular irritation testing and its improvement within the limits of OECD test guideline 437

Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.     

Evaluation of a rapid immunochromatographic diagnostic test for the detection of rabies from brain material of European mammals

The surveillance of rabies relies on investigations conducted on dead suspected animals or animals showing clinical signs suggestive of rabies. An immunochromatographic method based on lateral flow principle has been evaluated against a collection of brain samples mainly of European mammals including bats. The performance of this new test has been compared to the conventional gold standard methods: the fluorescent Antibody Test (FAT) and the Rapid Tissue Culture Infection Test (RTCIT). This test enabled the detection of various rabies strains belonging to rabies species 1, 5, 6 and 7 and demonstrated an overall specificity of 100% and a sensitivity of more than 88% when compared to FAT and RTCIT. A total agreement between the Rapid Immunochromatographic Diagnostic Test and conventional technique results have been obtained for European bat samples.