Approximate lethal dose versus median lethal dose in acute toxicity testing of pharmaceuticals

The relation between approximate lethal doses (ALD, i. e. the lowest dose at which mortality occurs) and the corresponding median lethal doses (LD₅₀) was investigated in 231 acute toxicity studies in mice and rats. The ALD values were divided into four classes (<5 mg/kg, 5–50 mg/kg, 50–500 mg/kg, 500–2000 mg/kg) and the LD₅₀/ALD factors were calculated. In intravenous studies the LD₅₀ values were higher than the ALD values by mean factors of 1.27–1.61 in mice and 1.25–2.84 in rats. In oral studies the LD₅₀ values were higher by mean factors of 1.46–2.5 in mice and 1.59–2.1 in rats. Only in 20 cases (8.7%) did the LD₅₀ values differ by factors higher than 2.     

The dermal acute toxic class method: test procedures and biometric evaluations

A dermal acute toxic class (ATC) method is presented with the use of significantly fewer animals in comparison with the classical dermal 50% lethal dose (LD₅₀) test. The principle of the dermal ATC method is based on the oral and the inhalation ATC method. The method was developed for three fixed starting doses. Depending on the dermal LD₅₀, the slope, the classification system and the starting dose on average 40 to 90% fewer animals will be used in comparison to at least 30 animals with the dermal LD₅₀ test. The method was biometrically evaluated by using the Probit model for dose-response relationships. At present, there are eight different international classification systems based on dermal LD₅₀ values. The test procedures and the calculations of the classification probabilities demonstrate that the dermal ATC method is a reliable alternative to the dermal LD₅₀ test with the use of significantly fewer animals. Classification probabilities are presented for all classification systems currently in use, and expected numbers of experimental and of moribund/dead animals are demonstrated for the system of chemicals in the European Union for all three starting doses. The conclusion is justified that, similarly to the inhalation ATC method, there is no need to validate the dermal ATC method with the use of experimental animals. Received: 24 June 1998 / Accepted: 7 September 1998     

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.     

Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals: I. Alternative toxicity measures as an estimator of carcinogenic potency

Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to identify alternative toxicity measures that may be used as surrogates for carcinogenic potency. Alternative toxicity measures for carcinogenic potency currently being used in the literature include lethal dose (dose that kills 50% of a study population [LD₅₀]), lowest-observed-adverse-effect-level (LOAEL) and maximum tolerated dose (MTD). The purpose of this study was to investigate the correlation between tumor dose (TD₅₀) and three alternative toxicity measures as an estimator of carcinogenic potency. A second aim of this study was to develop a Classification and Regression Tree (CART) between TD₅₀ and estimated/experimental predictor variables to predict the carcinogenic potency of new chemicals. Rat TD₅₀s of 590 structurally diverse chemicals were obtained from the Cancer Potency Database, and the three alternative toxicity measures considered in this study were estimated using TOPKAT^®, a toxicity estimation software. Though poor correlations were obtained between carcinogenic potency and the three alternative toxicity (both experimental and TOPKAT) measures for the CPDB chemicals, a CART developed using experimental data with no missing values as predictor variables provided reasonable estimates of TD₅₀ for nine chemicals that were part of an external validation set. However, if experimental values for the three alternative measures, mutagenicity and logP are not available in the literature, then either the CART developed using missing experimental values or estimated values may be used for making a prediction.     

硫辛酸对酒石酸锑钾解毒作用的研究

本文研究了国产硫辛酸对酒石酸锑钾的解毒作用.结果证明硫辛酸对酒石酸锑钾的急性及亚急性毒性均有非常显著的解毒作用.小白鼠腹腔注射酒石酸锑钾后,立即以130毫克/公斤的硫辛酸灌胃或皮下注射,可分别使酒石酸锑钾的急性LD₅₀提高1.6和1.9倍.在急性试验中,酒石酸锑钾为LD₉₅时,2-3分子硫辛酸可对抗1分子酒石酸锑钾;在亚急性试验中,酒石酸锑钾为LD₅₀时,1分子硫辛酸即可对抗1分子酒石酸锑钾.腹腔注射酒石酸锑钾LD₅₀后3小时皮下注射硫辛酸,可完全保护小白鼠免于死亡.硫辛酸性质稳定,口服吸收良好,在锑剂中毒出现症状后仍然有效,值得在临床试用.     

Toxicological studies on malachite green: A triphenylmethane dye

The oral LD₅₀ for malachite green oxalate was found to be 275 mg/kg in rats while the approximate lethal dose for NMRI mice was 50 mg/kg. No systemic effects were seen after dermal application of 2,000 mg/kg. Repeated administration in the diet for 28 days to rats produced only minor changes in serum urea and aspartate aminotransferase levels. The rats at the highest dose level showed decreased weight gain and appeared clinically to have elevated motor activity. No sex differences were observed in either acute or prolonged experiments. In accord with human experience malachite green was irritating to mucous membranes, but no effects were seen on intact skin nor was it shown to be sensitizing. It was found to be a mutagen in the Salmonella/microsome test after metabolic activation but without clastogenic activity when tested at maximally tolerated levels in mice in the micronucleus test.     

Toxicity studies of cremophor-free paclitaxel solid dispersion formulated by a supercritical antisolvent process

To evaluate the acute toxicity of a paclitaxel solid dispersion formulation, single dose studies in ICR mice were carried out for injectable excipients, paclitaxel solid dispersion powder, and Taxol®. In the dose range of excipients used for preparing paclitaxel solid dispersion, each excipient was clinically safe, and the LD₅₀ for exicipients was higher than 2,000 mg/kg for both males and females. In this study, there were no remarkable clinical signs or deaths related to paclitaxel solid dispersion even at doses up to 160 mg/kg of paclitaxel. But Taxol® resulted in clinical signs when it contained more than 30 mg/mL paclitaxel. The LD₅₀ for paclitaxel solid dispersion was above 160 mg/kg and the LD₅₀ for Taxol® was 31.3 mg/kg, more than 5 times lower than that of paclitaxel solid dispersion. However, paclitaxel solid dispersion could not be administered i.v. at a dose exceeding 160 mg/kg, because of high viscosity. To evaluate the nephrotoxicity of paclitaxel solid dispersion, plasma level of creatinine and kidney weight were measured and compared to Taxol®. At the doses administered, paclitaxel solid dispersion did not change creatinine clearance, while Taxol® killed all animals at doses >15 mg/kg. To investigate membrane damage when paclitaxel formulations were injected, hemolytic activity was determined for different concentrations. Paclitaxel solid dispersion showed about 10% hemolytic activity, whereas Taxol® showed about 40% hemolytic activity when it contained 2 mg of paclitaxel. Comparisons with the LD₅₀ value, nephrotoxicity, and hemolytic activity of Taxol® suggested that Cremophor-free paclitaxel solid dispersion as an injectable formulation is a promising approach to increasing the safety and clinical efficacy of paclitaxel for treatment of cancer.     

LD50 - its value for the pharmaceutical industry in safety evaluation of drugs.

Abstract Does the pharmaceutical industry perform LD₅₀-determinations in animals just because it is required by the authorities or are there any scientific benefits from counting dead animals and calculating and index of lethal toxicity? This is an important question when discussing LD₅₀ and possible alternatives. We will try to answer this question by presenting data and some views collected during almost ten years at a Swedish pharmaceutical company. We will describe how we have made use of the LD₅₀-values in the safety evaluation process. We will compare LD₅₀-values with the dose levels used in longterm toxicity both after single or repeated administration and with therapeutical dose levels in man for different classes of drugs. These data will enable us to demonstrate the value of the LD₅₀-determinations. As we are of the opinion that the LD₅₀-value itself has a limited value for the total safety evaluation of drugs we will look into the possibility of replacing the LD₅₀-determination with something else as an indication of lethal toxicity. In order to minimize the number of animals used and the number of animals dying because of dosing in studies on lethal toxicity we will try to support a proposal to use the maximal nonlethal dose (MNLD) as an indication of lethal toxicity in small animals.     

Toxicity study of Vernonia cinerea

The methanol extract of Vernonia cinerea Less (Asteraceae), which exhibited antimicrobial activity, was tested for toxicity. In an acute toxicity study using mice, the median lethal dose (LD₅₀) of the extract was greater than 2000?mg/kg, and we found no pathological changes in macroscopic examination by necropsy of mice treated with extract. As well as the oral acute toxicity study, the brine shrimp lethality test was also done. Brine shrimp test LC₅₀ values were 3.87?mg/mL (6?h) and 2.72?mg/mL (24?h), exhibiting no significant toxicity result. In conclusion, the methanol extract of V. cinerea did not produce toxic effects in mice and brine shrimp.     

Correlation between cytotoxicity in vitro and LD50-values.

Abstract Previous studies of the correlation between cytotoxicity in vitro and LD₅₀ in rodents have shown a good correspondence for small groups of substances, such as antibiotics, metals, and solvents, but a poor correspondence for other small groups of substances, such as drugs and pesticides, as well as for larger groups of heterogeneous chemicals. The generally poor correlation may be the result of the joint operation of three factors: 1. The existence of organ-specific toxicity. 2. The pharmacokinetics in whole animals. 3. Methodological problems disturbing a basically equivalent in vitro/in vivo toxicity. We have made further studies of the correlation for altogether 95 drugs. The type of human and mouse systemic lethal poisoning was compared with the cytotoxicity divided by the i.v. LD₅₀ in the mouse and approximate i.v. LD in man, called Specific Indices (SI_1d). A minority of the drugs had an organ-specific lethal action which was corresponding to high SI_1d of 100 or more for all drugs. A majority of the drugs (80%) had a known or possible lethal interference with basal functions of animal and human tissues correlated to low SI_1d of 1–10. At lethal dosage most of the latter drugs induce a non-specific CNS depression, which was interpreted as the systemic reaction to basal cytotoxicity of chemicals reaching brain cells. Tissue culture tests of cytotoxicity seem to be relevant for the lethal action of most chemicals in man. Comparisons of in vitro/in vivo toxic dosage (SI_1d) and concentrations might be used to screen the selectivity of lethal action of chemicals, and to select chemicals interfering with basal cell functions for further studies in vitro. It will be difficult to replace animal testing by cell tests in toxicity evaluation because of the organ-specific toxicity and characteristic pharmacokinetics of many substances. However, initial standard in vitro cytotoxicity tests combined with subsequent approximative LD₅₀ tests coupled to pharmacokinetic studies in the animals could produce data discriminating organ-specific from basal cell toxicity. The toxic mechanisms of the probable majority of common chemicals with a basal cytotoxicity at lethal dosage could then be analysed in tissue culture, which would increase the precision of analysis, as well as saving animal lives.     

Toxic effects of methylcyclopentadienyl manganese tricarbonyl (MMT) in rats: Role of metabolism

The toxicity of methylcyclopentadienyl manganese tricarbonyl (MMT) has been investigated in relation to its in vivo biotransformation in the rat. The LD₅₀ dose of MMT was found to be 50 mg/kg for oral administration and 23 mg/kg for ip administration. Death appeared to be caused by severe pulmonary hemorrhagic edema. Histological studies of MMT-treated animals revealed pathologic changes in lungs, liver, and kidney. Phenobarbital pretreatment protected rats from the lethal effects of 2.5 times the LD₅₀ dose of MMT, it shifted the site of tissue injury from the lungs to the liver, and it caused a doubling of the rate of biliary excretion of MMT metabolites. The possibility is discussed that MMT per se is toxic without bioactivation, and that the protective effect of phenobarbital pretreatment is due to a first-pass effect preventing toxic concentrations of orally administered MMT from reaching the systemic circulation.     

Solid lipid nanoparticles reduce systemic toxicity of docetaxel: Performance and mechanism in animal

Abstract

Nanotechnology presents great potential for increasing efficacy of docetaxel while reducing side-effects and toxicity. However, in vivo toxicity of nano-formulation of docetaxel has not been systemically investigated yet. Herein, the new docetaxel-loaded solid lipid nanoparticles (DSNs) were prepared, and systemic toxicity of DSNs in different animals was comprehensively investigated. The experimental results showed that no allergenicity and vascular irritation were induced by DSNs at the highest drug concentration of clinical infusion. The maximum tolerated dose (MTD) of DSNs was as high as 400 mg/kg in mice while the medial lethal dose (LD₅₀) of Taxotere was 149.31 mg/kg. The long-term toxicity of DSNs compared with Taxotere in beagle dogs by intravenous infusion weekly for four weeks showed that the administration of Taxotere at 1 mg/kg brought about severe signs of toxicity such as skin flushing, vocalization and salivation. However, no abnormal reactions appeared on animals treated with DSNs at dose of 4 mg/kg. At the same dose level, DSNs induced more minor decreases in body weight gains, slighter hemotoxicity (changes in some clinical hematology and biochemistry parameters), cardiac toxicity, hepatotoxicity and myelosuppression than Taxotere. These results could provide an important reference for developing the novel delivery system of docetaxel.     

Delayed neuropathy by the organophosphorus nerve agents soman and tabun

The organophosphorus nerve agents soman and tabun were tested in the hen at doses 120–150 times higher than their acute LD₅₀, as it was assumed that these doses would produce delayed neuropathy. The animals were protected against the acute lethal effect of these agents by pretreatment with atropine, physostigmine, diazepam, and the oxime HI-6 or obidoxime.The surviving animals were followed for 30 days and the occurrence of delayed neuropathy was clinically diagnosed. Soman produced severe delayed neuropathy at a dose of 1.5 mg/kg, a dose which produced acute lethality in five animals out of six. Tabun elicited very mild neuropathic symptoms in one animal out of two at a dose of 6 mg/kg given on 2 consecutive days. Delayed neuropathy was not seen in the hens that survived the acute toxicity of a single dose of tabun, 12 mg/kg (three out of six) or 15 mg/kg (two out of six).     

A safety study on single intravenous dose of tetrachloro-diphenyl glycoluril [iodogen] dissolved in dimethyl sulphoxide (DMSO)

Abstract

1. Iodogen (tetrachloro-diphenyl glycoluril) dissolved in DMSO (dimethyl sulphoxide) appears indispensable in radioiodination of hypericin for a new anticancer strategy. We studied the safety of intravenously administered iodogen/DMSO in mice (n?=?132).

2. Median lethal dose (LD₅₀) of iodogen/DMSO was determined with doses of 40.0, 50.0, 55.0, 60.0, 65.0 and 70.0?mg/kg. Next, toxicity of iodogen/DMSO at 30.0?mg/kg was evaluated using saline and DMSO as controls. Changes in behaviour, body weight and serum biochemistry were evaluated. Histopathology of lungs, heart, liver and kidney was performed.

3. LD₅₀ values of iodogen/DMSO were 59.5?mg/kg (95% confidence limits (CI): 54.1–65.4?mg/kg) and 61.0?mg/kg (95%CI: 56.2–66.2?mg/kg) for female and male mice, respectively. Similar to that of control groups, no animal deaths were encountered after iodogen/DMSO administration at 30.0?mg/kg. Body weights over 24?h were not altered in all groups, but significantly higher in iodogen/DMSO and DMSO groups (p?<?0.05) 14?d post-injection. Blood urea nitrogen and alkaline phosphatase increased (p?<?0.05) in iodogen/DMSO group without clinical symptoms. No pathologies were found by gross and microscopic inspection.

4. A single dose of iodogen/DMSO up to 30.0?mg/kg, over 3000 times the dose in potential human applications, appears safe, with an LD₅₀ doubling that dose in mice.     

The acute oral toxicity of isomeric monobutylamines in the adult male and female rat

The acute oral LD₅₀ values of n-butylamine, isobutylamine, sec.-butylamine, and tert.-butylamine were determined in both male and female Sprague-Dawley CD rats. Signs of toxicity observed after single oral doses of the monobutylamines included sedation, ataxia, nasal discharge, gasping, and salivation followed by convulsions and death at the higher dose levels. Gross pathological examination of animals that died after the monobutylamine treatment revealed pulmonary edema. The LD₅₀ values for the monobutylamines were calculated by the probit method of D. J. Finney (1971, Probit Analysis, 3rd ed., Cambridge Univ. Press, Cambridge). No significant sex-related differences were noted. The 14-day, po single-dose LD₅₀ values (mg/kg body wt) were: n-butylamine, male 365.4, female, 382.7; isobutylamine, male, 224.4, female, 231.8; sec.-butylamine, male, 157.5, female, 146.8; and tert.-butylamine, male, 82.3, female, 78.1.     

In vivo toxic and lethal cardiovascular effects of a synthetic polymeric 1,3-dodecylpyridinium salt in rodents

APS12-2 is one in a series of synthetic analogs of the polymeric alkylpyridinium salts isolated from the marine sponge Reniera sarai. As it is a potential candidate for treating non small cell lung cancer (NSCLC), we have studied its possible toxic and lethal effects in vivo. The median lethal dose (LD₅₀) of APS12-2 in mice was determined to be 11.5 mg/kg. Electrocardiograms, arterial blood pressure and respiratory activity were recorded under general anesthesia in untreated, pharmacologically vagotomized and artificially ventilated rats injected with APS12-2. In one group, the in vivo effects of APS12-2 were studied on nerve-evoked muscle contraction. Administration of APS12-2 at a dose of 8 mg/kg caused a progressive reduction of arterial blood pressure to a mid-circulatory value, accompanied by bradycardia, myocardial ischemia, ventricular extrasystoles, and second degree atrio-ventricular block. Similar electrocardiogram and arterial blood pressure changes caused by APS12-2 (8 mg/kg) were observed in animals pretreated with atropine and in artificially ventilated animals, indicating that hypoxia and cholinergic effects do not play a crucial role in the toxicity of APS12-2. Application of APS12-2 at sublethal doses (4 and 5.5 mg/kg) caused a decrease of arterial blood pressure, followed by an increase slightly above control values. We found that APS12-2 causes lysis of rat erythrocytes in vitro, therefore it is reasonable to expect the same effect in vivo. Indeed, hyperkalemia was observed in the blood of experimental animals. Hyperkalemia probably plays an important role in APS12-2 cardiotoxicity since no evident changes in histopathology of the heart were found. However, acute lesions were observed in the pulmonary vessels of rats after application of 8 mg/kg APS12-2. Predominant effects were dilation of interalveolar blood vessels and lysis of aggregated erythrocytes within their lumina.     

An accurate method for estimating an approximate lethal dose with few animals,tested with a Monte Carlo procedure

A reliable but not necessarily precise indication of the toxicity of a chemical product is frequently needed for the determination of its class of toxicity. Estimations of the LD₅₀ carried out for this purpose often have a precision which is higher than necessary and so is the number of laboratory animals used. Alternative methods estimating an approximate lethal dose (ALD) have been proposed, but too little is known about their accuracy and precision. The method of Deichmann and LeBlanc (1943) for estimating an ALD has a systematic error, dependent upon the magnitude of the unknown variance of the log tolerance. A new method was developed in which this systematic error was removed. Its performance was tested in a model with Monte Carlo techniques. The model is based on the log-normal distribution of individual tolerance, i.e. the lowest dose that is lethal for an individual of the species under study. A hypothetical substance was created with a mean tolerance between 1 and 5,000 mg per kg and a standard deviation of log tolerance between 0.1 and 1.5 (in natural logarithms). This substance was then subjected to a sequential test, by repeatedly drawing a random element from the population of normally distributed log tolerance values and testing whether this element is smaller or greater than the dose administered according to the method's protocol. The method of Lorke (1983) was tested with a similar simulation model. In series of 100 simulations no systematic error was found. For a standard deviation of log tolerance exceeding 0.85 the new method was less precise than that of Lorke, but for smaller values the new method was more precise; it required on average less than ten animals, against 13 required in Lorke's method.     

Acute lead poisoning of the pigeon induced by single,intraperitoneal administration of lead acetate

A single dose of lead acetate (either 30 mg/kg or 150 mg/kg) was intraperitoneally (i.p.) adminstered to adult feral pigeons, Columba livia var and the effects of calcium disodium ethylenediamine tetraacetate (CaNa₂EDTA), (0, 150, 300, 600 mg/kg), administered i.p. twice a week in the ensuing period were observed. Lead acetate caused dose related mortality and decreases in weight, hematocrit and -aminolevulinic acid dehydratase activity (ALA-D). Acute toxicity of lead acetate in the pigeon, when given intraperitoneally, appeared approximately equivalent to that in the rat and mouse in terms of LD₅₀. Blood lead (blood Pb) levels observed during the lethal stage were five to ten times less than those reported for chronic oral lead poisoning in the pigeon. Biological implications of elevated levels of blood Pb observed in the feral pigeon in the urban Tokyo area are discussed. CaNa₂EDTA induced dose related recovery in ALA-D in 30 mg/kg group, and reduction of blood Pb levels in the group dosed with 150 mg/kg of lead acetate.     

A new approach to minimizing the number of animals used in acute toxicity testing and optimizing the information of test results

170 acute toxicity determinations, carried out in rats, mice, and guinea pigs after oral and parenteral administration during the past 5 years, using 5 /5 animals per dose, were evaluated with respect to the possibility of a reduction of animals necessary for obtaining LD₅₀ values with limits of confidence. Calculations were performed on existing data; no additional animal experiments were initiated for the purpose of this paper.For the majority of substances 3 /3 animals per dose would have sufficed for the determination of a LD₅₀ value with limits of confidence. Even when using 2 /2 animals per dose, in most cases sufficently acceptable LD₅₀ values can be determined, if the period of observation — conventionally 4 weeks — would be doubled. Using this procedure, 75% of the animals could be saved. The reduced number of animals and the labor saved thereby would allow for a more individualized observation of single animals thus optimizing the evaluation of acute toxicity testing.     

Acute and subchronic dermal toxicity of Vitex negundo essential oil

Vitex negundo is a common herb in different herbal formulation. The potential acute and sub-chronic dermal toxicities were evaluated as per OECD (Organization for Economic Cooperation and Development) guidelines 402 and 411, respectively. Both sexes of Wistar rats were exposed to Vitex negundo oil of 2000?mg/kg body weight for acute dermal toxicity, whereas in the dermal sub-chronic toxicity study, rats were exposed to Vitex negundo oil 250, 500 and 1000?mg/kg body weight, respectively, for five times a week for 90?d. In acute and sub-chronic toxicity studies, all animals were normal without any behavioral, serum biochemistry, hematology, necroscopical and histopathological changes. The no observed effect level (NOEL) and no observed adverse effect level (NOAEL) of Vitex negundo oil were 250 and 1000?mg/kg/day, respectively. Vitex negundo oil is under the category 5 (Unclassified) according to the Globally Harmonized System, with an LD₅₀ value of over 2000?mg/kg.