Anthracyclines.

The genotoxicity and carcinogenicity data from in vitro and in vivo studies conducted during preclinical safety assessment of doxorubicin (DOXO), epirubicin (EPI) and idarubicin (IDA), are reviewed. The genotoxicity assays included a) gene mutation in Salmonella typhimurium with 5 tester strains; b) gene mutation in the V79 mammalian (lung) cell line; c) chromosome aberrations in human lymphocytes cultured in vitro; and d) chromosome aberrations in mouse bone marrow cells after intravenous (i.v.) administration in vivo. The long-term toxicity studies in the rat included a) single dose administration (3 mg/kg DOXO, 3.6 EPI and 0.75 IDA) to female rats of two different age groups, i.e. younger (7 weeks old at dosing) and older (13 weeks old), followed by one-year observation; and b) multiple dose administration to male and female rats (7 weeks old at dosing), consisting of i.v. administration of 0.25, 0.5 and 1 mg/kg DOXO or EPI and 0.06, 0.125 and 0.25 mg/kg IDA, once every 3 weeks for 10 cycles, followed by 18 months of observation. The genotoxicity studies revealed activity in gene mutation assays in bacterial and mammalian cells, and in chromosome aberration assays in human lymphocytes in vitro and in mouse bone marrow in vivo. In the two long-term studies in the rat, only mammary tumors were present. This finding was expected and, according to the literature, can be considered as species specific and not directly compound-related. The lack of tumor induction at the usual target organs for DNA reactive compounds, which are almost the same as those considered as target organs in anthracycline-exposed animals, indicates that the type and the extent of DNA damage precludes stimulation for proliferation and induction of neoplasia. Although an epigenetic mechanism can be hypothesized, support for such a mechanism is lacking.

Chemical reagents as potential impurities of pharmaceutical products: investigations on their genotoxic activity.

The genotoxic activity of chemical reagents and intermediates as potential impurities of final pharmaceutical products have been investigated by the AFI Mutagenesis Study Group. A number of compounds employed in the synthesis of beta-lactam (12), quinolone (6), antiviral (3), and other drugs (11) were analyzed. The information reported in this article was mainly obtained experimentally in our laboratories. In addition, attempts were made to obtain reference data; however, these were available for only a few compounds. The genetic end-point taken into account was principally gene mutation in bacteria. All chemical reagents used in the synthesis of quinolones and antivirals were negative in the Ames test. As far as reagents employed in beta-lactam synthesis were concerned, genotoxic activity was shown by the alkylating agents bromomethanol acetate and chloromethanol acetate, by carbon disulfide, and by the different dimethylanilines. The other chemicals generically considered as involved in "other syntheses" did not induce gene mutation, except for 2,5-dibromopentyl acetate, which was positive in the Ames test. For this compound, as for the halogenated methanol acetates, genotoxic activity was expected in view of its alerting chemical structure.

Mutagenic contaminants in synthetic peptides obtained by an azide coupling.

Hormone-like peptides are, almost by definition, not mutagenic. It was, therefore, unusual to find that some batches of peptides synthesized by azide coupling were mutagenic in the Ames test. One of these peptides, eledoisin, showed mutagenic activity particularly in Salmonella typhimurium TA 1535 without metabolic activation. This activity was independent of the peptide purity determined by HPLC and a dose response relationship was observed at concentrations over the solubility limit of the peptide in the assay medium. We therefore suggested that the mutagenic effect might be due to the presence of chemically undetectable, water-soluble impurities, which could be removed by counter-current distribution. If, however, the same final coupling was carried out by the mixed anhydride procedure, no mutagenic activity was observed. Consequently, we considered that the mutagenicity detected was due to traces of hydrazoic acid salts arising during azide formation in the coupling step. In fact only the product of the coupling reaction between the pivotal intermediates was mutagenic.

Micronuclei and nuclear anomalies induced in the gastro-intestinal epithelium of rats treated with formaldehyde.

The induction of micronuclei and nuclear anomalies in cells of the gastro-intestinal epithelium of rats treated per os with formaldehyde (200 mg/kg) was assessed in comparison with N-methyl-N'-nitro-N-nitrosoguanidine as a positive standard. Formaldehyde and N-methyl-N'-nitro-N-nitrosoguanidine both increased micronuclei and nuclear anomalies in almost all tissues analysed (stomach, duodenum, ileum and colon) though with different patterns and to different extents, reflecting different potency and specificity of target. In the case of formaldehyde these effects were observed in conjunction with signs of severe local irritation. This assay can be employed to detect the genotoxic potential of chemicals in vivo directly on target cells in the proximity of the administration site, thus reducing the likelihood of false-negative results.

DNA synthesis, mitotic index, drug-metabolising systems and cytogenetic analysis in regenerating rat liver. Comparison with bone marrow test after 'in vivo' treatment with cyclophosphamide.

Rat-liver cells can be used to reveal "in vivo" clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54-66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione-S-epoxide transferase were chosen as markers. All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed. DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22-25 and 28-31 h after partial removal of the liver. The sensitivity to clastogenic damage induced by "in vivo" treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure). The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.