Statistical tools for analysing the data obtained from repeated dose toxicity studies with rodents: a comparison of the statistical tools used in Japan with that of used in other countries.

In the present study, an attempt was made to compare the statistical tools used for analysing the data of repeated dose toxicity studies with rodents conducted in 45 countries, with that of Japan. The study revealed that there was no congruence among the countries in the use of statistical tools for analysing the data obtained from the above studies. For example, to analyse the data obtained from repeated dose toxicity studies with rodents, Scheffé's multiple range and Dunnett type (joint type Dunnett) tests are commonly used in Japan, but in other countries use of these statistical tools is not so common. However, statistical techniques used for testing the above data for homogeneity of variance and inter-group comparisons do not differ much between Japan and other countries. In Japan, the data are generally not tested for normality and the same is true with the most of the countries investigated. In the present investigation, out of 127 studies examined, data of only 6 studies were analysed for both homogeneity of variance and normal distribution. For examining homogeneity of variance, we propose Levene's test, since the commonly used Bartlett's test may show heterogeneity in variance in all the groups, if a slight heterogeneity in variance is seen any one of the groups. We suggest the data may be examined for both homogeneity of variance and normal distribution. For the data of the groups that do not show heterogeneity of variance, to find the significant difference among the groups, we recommend Dunnett's test, and for those show heterogeneity of variance, we recommend Steel's test.

Do we need to examine the quantitative data obtained from toxicity studies for both normality and homogeneity of variance?

Most of the statistical techniques used to evaluate the data obtained from toxicity studies are based on the assumption that the data show a normal distribution and homogeneity of variance. Literature review on toxicity studies on laboratory animals reveals that in most of the cases homogeneity of variance alone is examined for the data obtained from these studies. But the data that show homogeneity of variance need not always show a normal distribution. In fact, most of the data derived from toxicity studies, including hematological and biochemical parameters show a non-normal distribution. On examining normality of data obtained from various toxicity studies using different normality tests, we observed that Shapiro-Wilk test is more appropriate than Kolmogorov-Smimov test, Lilliefors test, the normal probability paper analysis and Chi square test. But there are situations, especially in the long-term toxicity studies, where normality is not shown by one or more than one of the dosage groups. In this situation, we propose that the data maybe analyzed using Dunnett multiple comparison test after excluding the data of the groups that do not show normality However, the biological relevance of the excluded data has to be carefully scrutinized. We also observed that the tendency of the data to show a normal distribution seems to be related to the age of the animals. Present paper describes various tests commonly used to test normality and their power, and also emphasizes the need of subjecting the data obtained from toxicity studies to both normality and homogeneity tests. A flow chart suggesting the statistical techniques that maybe used for both the types of data showing a normal or non-normal distribution is also proposed.

Evaluation of statistical tools used in short-term repeated dose administration toxicity studies with rodents.

In order to know the different statistical tools used to analyze the data obtained from twenty-eight-day repeated dose oral toxicity studies with rodents and the impact of these statistical tools on interpretation of data obtained from the studies, study reports of 122 numbers of twenty-eight-day repeated dose oral toxicity studies conducted in rats were examined. It was found that both complex and easy routes of decision trees were followed for the analysis of the quantitative data. These tools include Scheffe's test, non-parametric type Dunnett's and Scheffe's tests with very low power. Few studies used the non-parametric Dunnett type test and Mann-Whitney's U test. Though Chi-square and Fisher's tests are widely used for analysis of qualitative data, their sensitivity to detect a treatment-related effect is questionable. Mann-Whitney's U test has better sensitivity to analyze qualitative data than the chi-square and Fisher's tests. We propose Dunnett's test for analysis of quantitative data obtained from twenty-eight-day repeated dose oral toxicity tests and for qualitative data, Mann-Whitney's U test. For both tests, one-sided test with p=0.05 may be applied.

Screening of hepatoprotective effect of a herbal mixture against CCl4 induced hepatotoxicity in Swiss albino mice.

The hepatoprotective potential of a herbal mixture was evaluated against CCl4 induced liver injury in Swiss albino mice. Liv 52, a commercially available polyherbal hepatoprotective drug was evaluated for comparison. The potential toxicity of the above herbal hepatoprotective agents was also compared. It was observed that there was a reduction in the enzyme biomarkers (Aspartate and Alanine Transaminase) of liver injury in the herbal mixture treated groups, which was similar to the reduction initiated by Liv 52. An increase in glutathione was observed in the herbal mixture treated groups and it was assumed that the herbal mixture protects the liver by virtue of its antioxidant nature along with high regeneration initiation potential. From the study it is also concluded that the herbal mixture is safer than Liv 52.

Hypolipidemic effect of Coriandrum sativum L. in triton-induced hyperlipidemic rats.

In the biphasic model of triton-induced hyperlipidemia, C. sativum at a dose of 1g/kg body weight reduced cholesterol and triglycerides levels in both synthesis and excretory phases in rats, and the results were comparable with that of Liponil, a commercially available herbal hypolipidemic drug. The results suggest that coriander decreases the uptake and enhances the breakdown of lipids. From the study it can be assumed that coriander has the potential to be popularized as a household herbal remedy with preventive and curative effect against hyperlipidemia.

Formulation and in vivo evaluation of niosome-encapsulated daunorubicin hydrochloride.

The central aim of this present study was to modify the reverse evaporation process, such that an enhanced entrapment, with increased storage stability and prolonged release, could be achieved, and to translate these advantages to increased therapeutic efficacy of daunorubicin hydrochloride on Dalton's ascitic lymphoma. Niosomes prepared exhibited entrapment efficiency 20% higher than theoretically possible by the reverse evaporation process. The niosomes were found to be very stable at a storage temperature of 4 degrees C for a duration of three months. Even the drug leakage was restricted to just 10%. The in vivo studies suggested a prolonged release of 20 hr. Niosomal daunorubicin hydrochloride exhibited an enhanced anti-tumor efficacy when compared to free drug. The niosomal formulation was able to destroy the Dalton's ascitic lymphoma cells in the peritoneum within the third day of treatment, while free drug took around six days and the process was incomplete. The hematological studies also prove that the niosomal formulation was superior to free drug treatment. An enhanced mean survival time was achieved by the niosomal formulation that finally substantiates the overall efficacy of the niosomal formulation. This study suggests that the multilamellar vesicles obtained by the presently utilized reverse evaporation process resulted in vesicles that resisted the immediate lysis in the Kupffer cells, whereby a prolonged drug concentration was achieved which enhanced the cell lysis. But the major factor responsible for the quicker onset of action could be the increased permeability of the niosomes into the cell membrane and the cytoplasm of the Dalton's ascitic lymphoma cells.

Implications of fluoride--an endless uncertainty.

Fluorosis has become pandemic and it has assumed global status in the public health point of view. The paper deals with the health issues pertaining to fluorosis with special emphasis in the Indian context. It explains the equivocal evidences of fluoride-related problems and discusses the control measures of fluorosis. In general, it reveals how fluoride is useful at very low concentrations and at the same time brings forth manifestation of clinical abnormalities at higher concentrations that are the basis of the principles of toxicology.

Biochemical evaluation of multiple herbal treatments in alloxan-induced diabetes in Wistar rats.

Alloxan-induced diabetic rats were treated with multiple herbal preparation besides a control group receiving distilled water. The levels of glucose and alkaline phosphatase (ALP) increased abnormally in the alloxan treated group and the same were normalized upon treatment with the herbal preparation. The levels of blood urea nitrogen (BUN), alanine aminotransferase (ALT), protein and albumin in all groups remained unaltered. However, weekly body weight gain which got significantly altered in the alloxan-treated group was normalized by treatment with the herbal preparation. On the whole, a profound hypoglycemic effect was observed by the multiple herbal treatment in the diabetic rats.