Comparison of the up-and-down method and the fixed-dose procedure for acute oral toxicity testing

The acute oral toxicity data for 10 compounds, generated by using two alternative methods in rats, the up-and-down method and the fixed-dose procedure, were compared with those obtained from the classical LD50 test. In this evaluation, both alternative methods offered a reduction in animal use, while providing adequate information to rank the compounds according to the EEC classification for acute oral toxicity. In addition to the ranking, these alternative methods also provided useful information on signs of toxicity and gross autopsy findings, although the results varied depending on the method used. Of the three methods, the up-and-down method required the fewest animals. Although the up-and-down method used only females, the LD50 values obtained were in good agreement with those obtained by the classical method, which used both sexes. It is concluded that the up-and-down method and the fixed-dose procedure are acceptable alternative methods to the classical LD50 test, and the choice of method depends on the type of toxicity information required.     

Statistical evaluation of the revised fixed-dose procedure

The fixed-dose procedure (FDP) was introduced as OECD Test Guideline 420 in 1992, as an alternative to the conventional median lethal dose (LD50) test for the assessment of acute oral toxicity (OECD Test Guideline 401). The FDP uses fewer animals and causes less suffering than the conventional test, while providing information on the acute toxicity to allow substances to be ranked according to the EU hazard classification system. Recently the FDP has been revised, with the aim of providing further reductions and refinements, and classification according to the criteria of the Globally Harmonized Hazard Classification and Labelling scheme (GHS). This paper describes the revised FDP and analyses its properties, as determined by a statistical modelling approach. The analysis shows that the revised FDP classifies substances for acute oral toxicity generally in the same, or a more stringent, hazard class as that based on the LD50 value, according to either the GHS or the EU classification scheme. The likelihood of achieving the same classification is greatest for substances with a steep dose-response curve and median toxic dose (TD50) close to the LD50. The revised FDP usually requires five or six animals with two or fewer dying as a result of treatment in most cases.     

Oral acute toxic class method: a successful alternative to the oral LD50 test

The oral acute toxic class method (ATC method) was developed as an alternative to replace the oral LD50 test. The ATC method is a sequential testing procedure using only three animals of one sex per step at any of the defined dose levels. Depending on the mortality rate three but never more than six animals are used per dose level. This approach results in the reduction of numbers of animals used in comparison to the LD50 test by 40-70%. The principle of the oral ATC method is based on the Probit model and it was first evaluated on a biometric basis before a national and subsequently an international ring study were conducted. The results demonstrated an excellent agreement between the toxicity and the animal numbers predicted biometrically and observed in the validation studies. The oral ATC method was adopted as an official test guideline by OECD in 1996 and was slightly amended in 2001. The ATC method has been successfully used in Germany and in 2003 >85% of all tests on acute oral toxicity testing was conducted as oral ATC tests. In member states of the European Union the ATC method is used in the range of 50% of all tests conducted. Meanwhile the oral LD50 test has been deleted by OECD, by the European Union and by the USA, making the use of alternatives to the oral LD50 test mandatory.     

Statistical evaluation of an acute dermal toxicity test using the dermal fixed dose procedure

The conventional method for the assessment of acute dermal toxicity (OECD Test Guideline 402, 1987) uses death of animals as an endpoint to identify the median lethal dose (LD50). A new OECD Testing Guideline called the dermal fixed dose procedure (dermal FDP) is being prepared to provide an alternative to Test Guideline 402. In contrast to Test Guideline 402, the dermal FDP does not provide a point estimate of the LD50, but aims to identify that dose of the substance under investigation that causes clear signs of nonlethal toxicity. This is then used to assign classification according to the new Globally Harmonised System of Classification and Labelling scheme (GHS). The dermal FDP has been validated using statistical modelling rather than by in vivo testing. The statistical modelling approach enables calculation of the probability of each GHS classification and the expected numbers of deaths and animals used in the test for imaginary substances with a range of LD50 values and dose-response curve slopes. This paper describes the dermal FDP and reports the results from the statistical evaluation. It is shown that the procedure will be completed with considerably less death and suffering than guideline 402, and will classify substances either in the same or a more stringent GHS class than that assigned on the basis of the LD50 value.     

A simple method for screening assessment of acute toxicity of chemicals

We proposed a simple method for screening assessment of acute oral and dermal toxicity using only three rats and mice of each sex at each dose level. Animals were first treated with chemicals at a dose of 2000 mg/kg and were carefully observed for compound-related morbidity and mortality. If none of the animals died, the following toxicity tests were suspended. If some of the animals died, toxicity tests at doses of 200 and 20 mg/kg were performed. The approximate LD₅₀ values calculated by this method showed little difference between two separate laboratories and were in good agreement with LD₅₀ values reported in the literature. Our toxicological data also showed that LD₅₀ values were about 2–2.5 times the MNLD (maximum non lethal dose) in acute oral and dermal toxicity. This meant that a chemical could be regarded as having an LD₅₀ of about 4000 mg/kg or higher when there was no mortality at the dose of 2000 mg/kg. A chemical with such low toxicity would not require further testing for lethal effects. Therefore, this simple method combining the fixed-dose procedure with the limit test is suitable for determination of approximate LD₅₀ values of chemicals and for screening for necessity for classical full LD₅₀ test using many animals.This work was supported by a grant from Ministry of Health and Welfare in Japan (No. 467 and 511)     

A simplified method to evaluate the acute toxicity of ricin and ricinus agglutinin

A brief method is described for the assessment of lethal toxicity of ricin and ricinus agglutinin using a modified LD(50) assay. With this test, it is possible to obtain an LD(50) using only 10 experimental animals. The equation for calculating the confidence limit of LD(50) is derived in this article. The LD(50) values obtained with the method were compared with data reported in the literature using classical methods. The results indicate that the simplified method for evaluating the acute toxicity of ricin and ricinus agglutinin gives reliable results. For scientific, economic and ethical reason the method is suggested to replace the traditional LD(50) assay for plant toxins.     

Dermal and inhalation acute toxic class methods: test procedures and biometric evaluations for the Globally Harmonized Classification System

The acute toxic class (ATC) methods were developed for determining LD(50)/LC(50) estimates of chemical substances with significantly fewer animals than needed when applying conventional LD(50)/LC(50) tests. The ATC methods are sequential stepwise procedures with fixed starting doses/concentrations and a maximum of six animals used per dose/concentration. The numbers of dead/moribund animals determine whether further testing is necessary or whether the test is terminated. In recent years we have developed classification procedures for the oral, dermal and inhalation routes of administration by using biometric methods. The biometric approach assumes a probit model for the mortality probability of a single animal and assigns the chemical to that toxicity class for which the best concordance is achieved between the statistically expected and the observed numbers of dead/moribund animals at the various steps of the test procedure. In previous publications we have demonstrated the validity of the biometric ATC methods on the basis of data obtained for the oral ATC method in two-animal ring studies with 15 participants from six countries. Although the test procedures and biometric evaluations for the dermal and inhalation ATC methods have already been published, there was a need for an adaptation of the classification schemes to the starting doses/concentrations of the Globally Harmonized Classification System (GHS) recently adopted by the Organization for Economic Co-operation and Development (OECD). Here we present the biometric evaluation of the dermal and inhalation ATC methods for the starting doses/concentrations of the GHS and of some other international classification systems still in use. We have developed new test procedures and decision rules for the dermal and inhalation ATC methods, which require significantly fewer animals to provide predictions of toxicity classes, that are equally good or even better than those achieved by using the conventional LD(50)/LC(50) methods. In order to cope with rather narrow dose/concentration classes of the GHS we have, as in our previous publications, combined the outcome of all results that can be obtained during testing for the allocation to one of the defined toxicity classes of the GHS. Our results strongly recommend the deletion of the dermal LD(50) and the inhalation LC(50) test as regulatory tests and the adoption of the dermal and inhalation ATC methods as internationally accepted alternatives.     

基因重组Kringle 5蛋白急性经口毒性试验研究

目的检测基因重组Kringle 5蛋白的急性经口毒性,为其临床应用提供医学毒理学依据。方法采用小鼠急性经口毒性试验并采用霍恩法测定半数致死量(LD50)。结果 1周内试验动物未见明显中毒症状及死亡发生,采用霍恩法测定LD50>10g/kg。结论基因重组Kringle 5蛋白属实际无毒级。     

A confirmatory study of the up-and-down method for acute oral toxicity testing

Ten materials have been tested in parallel both by the “classical” method for acute oral toxicity (LD50) and by the up-and-down method. Materials tested included laundry and dishwashing detergents, a shampoo, a flavor, potassium hydroxide, and caffeine. All testing was done in Sprague-Dawley rats. Excellent agreement was seen between the two methods. The classical method typically used 40 to 50 animals while the up-and-down method required only six to nine animals per material.     

Acute and oral subchronic toxicity of D-003 in rats

D-003 is a mixture of higher aliphatic primary acids purified from sugar cane wax (Saccharum officinarum) with cholesterol-lowering and antiplatelet effects experimentally proven. The present work reports the results of two studies investigating the acute and subchronic oral toxicity of D-003 in rats. Oral acute toxicity of D-003 (2000 mg/kg) was investigated according to the Acute Toxic Class (ATC) method (an alternative for the classical LD(50) test), which was performed in Wistar rats. The results obtained in this study defined D-003 oral acute toxicity as unclassified. In the subchronic study, rats of both sexes were orally treated with D-003 at 50, 200 and 1250 mg/kg for 90 days. At this time, animals were sacrificed. No evidence of treatment-related toxicity was detected during the study. Thus, data analysis of body weight gain, food consumption, clinical observations, blood biochemical, haematology, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. It is concluded that D-003 orally administered to rats was safe and that no drug-related toxicity was detected even at the highest doses investigated in both acute (2000 mg/kg) and subchronic (1250 mg/kg) studies.     

Up-and-down procedure (UDP) determinations of acute oral toxicity of nitroso degradation products of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used military explosive and soil and ground water contaminant of munitions manufacturing and artillery training sites, undergoes microbial nitroreductase metabolism to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Human occupational and accidental exposures to RDX, as well as acute oral exposures in rats, result in seizures, but little is known about the toxicity of the RDX degradation products. The main objective of the present study was to determine the oral LD50 of the most potent RDX N-nitroso product in female Sprague-Dawley rats using the recently validated up-and-down procedure (UDP). With only 26 rats, MNX was identified as the most potent metabolite and a maximum likelihood estimate of 187 mg kg(-1) (95% confidence interval 118-491 mg kg(-1)) for its LD50 was established and found equivalent to that of RDX determined with the same protocol. CNS toxicity, manifested as forelimb clonic seizures progressing to generalized clonic-tonic seizures, was the critical adverse effect. Further, confirmation of the UDP LD50 for MNX with a fixed-dose design enabled identification of 94 mg kg(-1) as the highest nonlethal dose. An ED50 of 57 mg kg(-1) was determined for neurotoxicity, while splenic hemosiderosis and decreased blood hematocrit and hemoglobin concentration occurred with a threshold at 94 mg kg(-1) in 14-day survivors. These studies, while providing new toxicity data necessary for the management of RDX-contaminated sites, illustrate the efficiency of the UDP for comparative acute toxicity determinations and its value in guiding further characterization of dose dependency of identified adverse effects.     

Significance of the LD50-test for the toxicological evaluation of chemical substances

The LD₅₀-test was developed in 1927 for the biological standardization of dangerous drugs. Then it was incorporated into the routine toxicological protocol of other classes of chemical compounds and is now part of practically all governmental guidelines which regulate toxicological testing of chemicals.For scientific, economic, and ethical reasons it is necessary to periodically reassess all toxicological test procedures, including the LD₅₀-test. Tests which are not optimal or that have become obsolete because of new scientific knowledge, must be changed or eliminated.The review of the LD₅₀-test shows that the precision of the procedure is dependent on the number of animals used. But even with large numbers of animals there are considerable variations of the test results, because the numerical value of the LD₅₀ is influenced by many factors, such as animal species and strain, age and sex, diet, food deprivation prior to dosing, temperature, caging, season, experimental procedures, etc. Thus, the LD₅₀ value cannot be regarded as a biological constant.Through standardization of the test animals and the experimental conditions the variability of the LD₅₀ determinations can be reduced but never fully eliminated.There are several tests with which an approximate LD₅₀ can be determined. These methods use fewer animals than the classical LD₅₀-test, but their precision and reproducibility are sufficient for most purposes of acute toxicity testing.Through incorporation of physiological, hematological, biochemical, pathological, and histopathological investigations in the simplified test procedures with small numbers of animals, it is possible to markedly increase the informational content of the results with regard to the toxicological spectrum and the target organs of toxicity. Such studies have already replaced the LD₅₀-test in large animals, such as dogs and monkeys. It is also desirable to replace the LD₅₀ in rodents with such a procedure.With pharmacologically inert compounds that have no acute effects with single administration the classical LD₅₀-test does not provide relevant toxicological results.For the prediction of the human lethal dose and for the prediction of the symptomatology of poisoning after acute overdosing in man the LD₅₀-test is of limited usefulness. An acute toxicity test with small numbers of animals combined with comprehensive studies of physiological functions, biochemical and histopathological examinations often provides more important information for emergency physicians and poison control centers.For the selection of doses to be used in subacute and chronic toxicity experiments the LD₅₀-test does not provide consistent and reliable results. A simple pilot experiment with few animals but repeated dosing gives more useful information.For the evaluation of special risks for the human newborn and infant the LD₅₀-test is poorly suited.For the appraisal of pharmacokinetic behavior and bioavailability the LD₅₀-test gives only semi-quantitative, often ambiguous information.In all cases where the acute toxicity testing is mainly concerned with the evaluation of toxicological potential of the test substances, the symptomatology following acute overdosing, and the knowledge of target organs of toxicity, the classical LD₅₀-test should be replaced by a more comprehensive short term test that can be done with small numbers of animals. The classical LD₅₀-test should only be permitted in those rare instances where a high precision of the LD₅₀ determination is indispensable.     

Alternative approaches in median lethality (LD50) and acute toxicity testing

The LD50 test was introduced by Trevan in 1927 for biological standardization of dangerous drugs. Since then, the LD50 has gained wide acceptance as a measure of acute toxicity of all types of substances. Recently, however, the LD50 test has been criticized as an unnecessary waste of resources. Therefore, efforts have been made to reduce the number of animals used in such tests and to avoid using this test unless required by regulations. A review of the literature has shown that a relatively small number of animals per dose level (5) and a small number of dose levels (2 or 3) are usually sufficient to calculate an LD50 and slope using moving average methods. In addition, one sex should suffice since large sex differences are seldom encountered. When a formal LD50 is not required, one of several approximate methods may be used to estimate the lethal dose. Future approaches include in vitro cytotoxicity methods and computer-based structure-activity models. The in vitro methods are still in an early stage of development and will require extensive validation before they are accepted by the toxicology community. In conclusion, when LD50 tests are required, the most economical approach should be used, without undue concern for statistical precision.     

Sirc-cvs cytotoxicity test: an alternative for predicting rodent acute systemic toxicity

An in vitro crystal violet staining method using the rabbit cornea-derived cell line (SIRC-CVS) has been developed as an alternative to predict acute systemic toxicity in rodents. Seventy-nine chemicals, the in vitro cytotoxicity of which was already reported by the Multicenter Evaluation of In vitro Toxicity (MEIC) and ICCVAM/ECVAM, were selected as test compounds. The cells were incubated with the chemicals for 72 hrs and the IC(50) and IC(35) values (microg/mL) were obtained. The results were compared to the in vivo (rat or mouse) "most toxic" oral, intraperitoneal, subcutaneous and intravenous LD(50) values (mg/kg) taken from the RTECS database for each of the chemicals by using Pearson's correlation statistics. The following parameters were calculated: accuracy, sensitivity, specificity, prevalence, positive predictability, and negative predictability. Good linear correlations (Pearson's coefficient; r>0.6) were observed between either the IC(50) or the IC(35) values and all the LD(50) values. Among them, a statistically significant high correlation (r=0.8102, p<0.001) required for acute systemic toxicity prediction was obtained between the IC(50) values and the oral LD(50) values. By using the cut-off concentrations of 2,000 mg/kg (LD(50)) and 4,225 microg/mL (IC(50)), no false negatives were observed, and the accuracy was 84.8%. From this, it is concluded that this method could be used to predict the acute systemic toxicity potential of chemicals in rodents.     

An alternative approach for the safety evaluation of new and existing chemicals, an exercise in integrated testing

Various in vitro and in silico methods without animals were applied to 10 substances listed on ELINCS with a complete VIIA base-set available at NOTOX. The hazard assessment for these substances was performed on basis of available non-animal data, QSAR, PBBK-modelling and additional, new in vitro testing was applied. Based on these data predictions on fish toxicity, acute toxicity, skin- and eye-irritation, sensitisation, and toxicity after repeated dosing were made. The predictions were compared with the outcome of the in vivo tests. Nine out of ten predictions on fish LC(50) proved to be correct. For skin- and eye-irritation 70% was predicted correctly. Sensitisation was predicted correctly for 7 out of 10 substances, but three false negatives were found. Acute oral toxicity (LD(50)) and repeated dose toxicity were less successful (5 out of 10 and 2 out of 10 correct predictions, respectively); application of the PBBK model proved successful. Acute dermal toxicity was predicted correctly in 9 out of 10 cases. In general an over-estimation of systemic toxicity was found, which can be explained by an over-prediction of cytotoxicity and worst case assumptions on absorption and binding to (plasma) proteins. This integrated approach leads to a 38% reduction of laboratory animals.     

The international validation study of the acute toxic class method (oral)

 An alternative to the oral LD₅₀ test, the acute toxic class (ATC) method (oral), was validated with 20 substances in an international collaborative study with nine laboratories in five countries. The ATC method is a stepwise procedure with the use of three animals per step. It has been designed with three fixed doses (25, 200 and 2000 mg/kg). In general, this testing is sufficient for allocation to the toxicity classes of the majority of the international classification systems currently in use. The selection of testing at additional fixed doses (5, 50 and 500 mg/kg) may be considered if further refinement is necessary or for specific allocation to those international classification systems with a cut-off value of 5 mg/kg. On average, two to four steps are necessary to complete a test. With the ATC method substances can be ranked in a similar or even better manner than with an LD₅₀ test but it uses up to 90% fewer animals, the average being 70% fewer. This also results in substantially fewer moribund/dead animals. The ATC method is based on biometric evaluations that, together with the experimental results, demonstrate that this method is a sensitive and reliable alternative to the LD₅₀ test. Received: 10 March 1995 / Accepted: 27 April 1995     

Comparison of acute toxicities of deoxynivalenol (vomitoxin) and 15-acetyldeoxynivalenol in the B6C3F1 mouse

The acute toxic effects of deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-ADON) were compared in the B6C3F1 female mouse after oral and intraperitoneal exposure. Using the abbreviated procedure of Lorke (Archs Toxicol. 1983, 54, 275), LD50 values for DON were estimated to be 78 mg/kg (oral) and 49 mg/kg (ip) whereas the LD50 values for 15-ADON were 34 mg/kg (oral) and 113 mg/kg (ip). Acute doses of these toxins resulted in extensive necrosis of the gastro-intestinal tract, bone marrow and lymphoid tissues, and focal lesions in kidney and cardiac tissue. The minimum doses required for these histopathological effects were consistent with LD50 estimations. The results indicate that 15-ADON was more or less toxic than DON depending on the route of administration. Risk assessments for DON should therefore consider the potential for 15-ADON occurrence and toxicity in food and feed.     

7种化合物细胞毒性与小鼠急性毒性的相关性研究

目的：为减少实验动物的使用,利用化学物质的体外细胞毒性数据对体内急性毒性进行预测。方法：MTT比色法检测7种新化学实体对CHL细胞的毒性作用,利用RC（Registry of Cytotox-icity）预测模型对急性毒性LD50值进行预测,并使用小鼠急性毒性上下法进行验证。结果：各化合物（1～7）细胞毒性IC50值分别为0.43、0.49、0.18、0.67、3.03、1.68、1.79mg/mL;根据RC预测模型,急性毒性LD50的预测值分别为2376.4、2478.3、1574.8、2087.6、4897.3、3331.8、3300.7mg/kg。经上下法检测,4号化合物的LD50值为1634.0mg/kg,其余6种化合物的LD50值均大于2000.0mg/kg。分别以预测值和实测值为依据对化合物毒性进行分级,二者相比,仅3号和4号化合物毒性分级略有差异,其它5种化合物的毒性分级基本一致。结论：体外细胞毒性数据可用来预测体内急性毒性,减少实验动物使用。     

拟原白头翁素A毒性试验研究

目的为了对拟原白头翁素Ａ进行初步毒理学安全性评价。方法采用大鼠急性经口和经皮毒性试验，大鼠皮肤和大白兔眼粘膜刺激试验，Ａｍｅｓ试验，小鼠骨髓微核试验，小鼠睾丸初级精母细胞染色体畸变试验。结果大鼠急性经口ＬＤ５０４．６７３９（ＩＣ４．０６１４～５．３７８７）ｇ·ｋｇ－１，经皮ＬＤ５０＞２．０ｇ·ｋｇ－１，根据化合物毒性分级为低毒；对大鼠皮肤无刺激作用，对大白兔眼粘膜有中等刺激；Ａｍｅｓ试验、小鼠骨髓微核试验和小鼠睾丸初级精母细胞染色体畸变试验阴性。结论拟原白头翁素Ａ为低毒、致突变试验阴性的化合物。