SFTG international collaborative study on in vitro micronucleus test IV. Using CHL cells.

In this report, are presented the results of an international collaborative study on the in vitro micronucleus assay, using CHL cells. Fourteen laboratories participated in this study which was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances, having different modes of action and at different levels were assessed in the in vitro micronucleus test, using a common protocol. Mitomycin C was used as a positive control. In order to help to define a standard protocol on CHL cells, short and long treatment periods followed by various recovery times, with or without cytochalasin B, were compared. After an evaluation of the acceptability of the assays, the tested chemicals were classified as negative, positive or equivocal. Mannitol and clofibrate were judged as negative in all treatment schedules. Bleomycin was positive in all the treatment schedules, with an increase in the number of micronucleated cells in both mononucleate and binucleate cells when using cytochalasin B. This was also shown for the aneugens colchicine, diethylstilboestrol and griseofulvin, as expected. Urethane was judged as equivocal only after long treatment with cytochalasin B, and negative in all other treatment schedules. In any case, no genotoxic compound would have been missed with schedules including a short and a long treatment time, whether the treatment was followed by a recovery period or not and whether cytochalasin B was used or not. Thus, these results show that CHL cells were suitable for accurately detecting clastogenic and aneugenic compounds of various types in the in vitro micronucleus test.

An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells.

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.

Muscle distribution of antimicrobial agents after a single intravenous administration to rats.

The purpose of this study was to evaluate the distribution of three fluoroquinolones (pazufloxacin, ciprofloxacin and ofloxacin) and a beta-lactam, ceftazidime in the tissue interstitial and intracellular spaces after a single intravenous administration to rats based on muscle microdialysis. The unbound concentration in the tissue interstitial fluid (C(isf,u)) after administration was estimated from the concentration in the dialysate by muscle microdialysis, the in vitro permeability rate constant, and the previously reported effective dialysis coefficient. The C(isf,u)s of pazufloxacin, ciprofloxacin, ofloxacin and ceftazidime in the muscle were close to their unbound concentrations in the venous plasma. These results were consistent with ones previously obtained at steady state. Based on these results, the total concentration in the tissue interstitial fluid (C(isf)) was calculated from the ratio of plasma protein binding, the plasma concentration, and previously reported interstitial-to-plasma albumin ratio in muscle of rats. The calculated C(isf) was compared with the muscle concentration (C(m)) obtained using the homogenized tissue. The C(isf) of ceftazidime was higher than the C(m). The C(isf) of pazufloxacin was found to be almost equal to its C(m). The C(isf)s of ciprofloxacin and ofloxacin were lower than their C(m)s with the exception of the values at 5 min after administration. These results indicate that ceftazidime is mainly distributed in the interstitial space of the muscle, that pazufloxacin is distributed equally in both the interstitial space and the tissue cells, and that ciprofloxacin and ofloxacin are mainly distributed in the tissue cells rather than the interstitial space.