The rat bone marrow micronucleus test: Statistical considerations on historical negative control data.

Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group "Statistics" within the German-speaking "Gesellschaft für Umwelt-Mutationsforschung e.V." (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified "laboratory" and "strain", but not "gender" as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.

Measurement of glomerular filtration rate in the conscious rat.

INTRODUCTION: Glomerular filtration rate (GFR) is an important parameter for studying drug-induced impairments on renal function in rats. The GFR is calculated from the concentration of creatinine and blood urea nitrogen (BUN) in serum and in urine, respectively. Following current protocols serum and urine samples must be taken from the same animal. Thus, in order to determine time-dependent effects it is necessary to use for each time point one separated group of animals. We developed a statistical test which allows analyzing the GFR from two different groups of animals: one used for repeated serum and the other one used for repeated urine analysis. METHODS: Serum and urine samples were taken from two different sets of rats which were otherwise treated identically, i.e. drug doses, routes of administration (per os or per inhalation) and tap water loading. For each dose group GFR mean, standard deviation and statistical analysis to identify differences between the dose groups were determined. RESULTS: After determination of the optimal time points for measurements, the effect on GFR of the three reference compounds, furosemide, hydrochlorothiazide and formoterol, was calculated. The results showed that the diuretic drugs furosemide and hydrochlorothiazide decreased the GFR and the antidiuretic drug formoterol increased the GFR, as counter regulation on urine loss or urine retention, respectively. DISCUSSION: A mathematical model and the corresponding algorithm were developed, which can be used to calculate the GFR, and to test for differences between groups from two separated sets of rats, one used for urine, and the other one for serum analysis. This new method has the potential to reduce the number of animals needed and to improve the quality of data generated from various groups of animals in renal function studies.