Acute and Genotoxic Profile of a Dimeric Impurity of Cefotaxime

Abstract

The manufacturing and storage of cefotaxime produces different impurities of various concentrations, which may influence the efficacy and safety of the drugs. Because no report of toxicity data is available on the impurities of cefotaxime, the present acute and genotoxicity studies were designed and conducted to provide the information for establishing the safety profile and qualification of the dimeric impurity. Histidine-requiring mutants of Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains, with or without metabolic activation (S-9), were used for point-mutation tests. Neither increase in numbers of revertants, indicative of mutagenic activity, nor inhibition of bacterial growth, indicative of cytotoxicity, was observed when the dimeric impurity of cefotaxime at concentrations of 0.62, 1.85, 5.56, 16.67, and 50 μg/plate was incorporated into plates containing S. typhimurium bacterial strains. Cultures of Chinese hamster ovary (CHO) cells at a cell density of 2 × 10⁵ cells per culture were exposed to the dimeric impurity of cefotaxime at the concentration of 11.25, 22.5, and 45 mg per culture, with or without metabolic activation, and harvested at 18 h after exposure. No chromosomal aberrations in the cultured mammalian cells were recorded. Acute intramuscular administration of the dimeric impurity of cefotaxime in Sprague-Dawley rats did not result in any clinical signs and gross pathological changes up to 2000 mg/kg-body weight. The results of these studies indicated that the dimeric impurity of cefotaxime is nonmutagenic in Ames test, nonclastogenic in vitro, and acutely nontoxic in rats.

Cefotaxime, an antibiotic of the cephalosporin group, is widely used against a large number of gram-positive and gram-negative bacteria and also microorganisms with resistance to β-lactamases. Cephalosporin structures have a 7-aminocephalosporanic acid nucleus with two variable side chains at the 3 and 7 positions of the ring. The sodium salt of 7-[2-(2-amino-4-thiazolyl)glyoxylamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 7²(z)-(O-methylo-xime), acetate (ester) (cefotaxime) (Figure 1) is used parenterally ( AHFS drug information 2002). The manufacturing and storing process of cefotaxime produces impurities of various concentrations. The presence of small amounts of impurities may influence the efficacy and safety of the drugs. Identification and quantification of impurities are required for the submission of registration/notification of dossiers to the regulatory authorities. Occasionally, the concentration of these impurities crosses the threshold limit and warrants establishing the safety pro-file (International Conference on Harmonization [ICH] 2000a, International Conference on Harmonization [ICH] 2000b). Six qualified impurities of cefotaxime are reported in Pharmeuropa, 2002. However, no toxicity data are available on the individual impurities. Quantitative computer-assisted toxicity prediction software “Topkat” was used for the prediction of toxicity of the dimeric impurity and the result obtained did not meet the validation criteria. Therefore, to provide the information for establishing the safety profile and qualification of the impurity, the present toxicity studies were designed and conducted.

MATERIALS AND METHODS

The dimeric impurity of cefotaxime was isolated and characterised using liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) (Penalvo et al. 1996).

The test substance (high-performance liquid chromatography [HPLC] purity 92.56%) was subjected to toxicity screening for the qualification of impurity as per the ICH recommendation (ANDAs impurities in drug products 1998; Impurities in new substances …in ANDAs 2000).

Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and TA102 were obtained from Bruce Ames Laboratory, Molecular and Cell Biology, University of California, and were checked for their viable counts and genotype characteristics. The reversion of histidine deficient auxotrophs of S. typhimurium was studied in the presence and absence of Aroclor 1254–induced Sprague-Dawley rat S-9 prepared by the method of Maron and Ames (1983). Fresh S-9 mix was prepared as per the method of Venitt, Crofton-Sleigh, and Forster (1990). Overnight broth cultures of tester strains having a cell density of (1–3) ×10⁹ cells per milliliter were used for preparation of bacterial lawns on minimal agar medium. Preliminary cytotoxicity evaluation was studied with and without metabolic activation in a single bacterial tester strain, TA100. As thinning of the background lawn and reduced number of histidine revertant colonies were observed at 50 μg/plate, this dose level was selected as the top dose. Subsequently, four lower concentrations (16.66, 5.55, 1.87, and 0.62 μg/plate) were prepared by serial dilution and 100 μl of the test solution was incorporated into plates containing S. typhimurium bacterial strains. Concurrent positive and negative (distilled water) controls were used in the study. 2-Amino anthracene (0.5 μg/plate) for strain TA1535, 2-aminoflourene (10 μg/plate) for strains TA97a, TA98, TA100, and Danthron (30 μg/plate) for strain TA102 were used as positive controls for the experiment carried out with S-9. In case of the experiment carried out without S-9, sodium azide (0.5 μg/plate) was used for strain TA1535, 4-nitroquinoline-N-oxide (0.5 μg/plate) for strains TA97a and TA98, and methyl methane sulphonate (0.5 μg/plate) for strains TA100 and TA102 as positive control. Sodium azide and 4-nitroquinoline-N-oxide were procured from Fluka Chemicals, Germany, and all other positive controls were obtained from Sigma, St. Louis, MO, USA. Experiments were performed twice (3 plates/dose/experiment). The plates were checked for their sterility, observed for a uniform lawn of auxotrophs (his⁻) and for the colonies of histidine revertants (his⁺). Histidine revertants were counted manually and the results are shown as the mean of three plates with standard deviation.

CHO cell line was obtained from National Cell Culture Supply, Pune, India, for in vitro chromosomal aberration studies. The obtained cells were fibroblastic, with modal chromosomal number of 2n = 22 and cell doubling time of 12 to 14 h. Monolayer cultures of 80% confluency were cultured at a cell density of 2.3×10⁵ cells per culture, and 24 h after culturing, were exposed to the test substance with and without Aroclor 1254–induced Sprague-Dawley rat S-9. As no precipitation and reduced mitotic index (24.61% and 14.13%) was recorded for dimeric impurity of cefotaxime at 45 mg/culture, dose levels of 45, 22.5, and 11.25 mg/culture were selected and exposed to cell cultures in duplicate (U.S. Environmental Protection Agency 1998). Concurrent positive (ethyl methane sulphonate [E. Merck, USA] without S-9 and benzo(a)pyrene with S-9 (Spectrochem Pvt., India) and negative control (distilled water) were used for the study. Cell cultures were incubated at 37^°C, harvested at 18 h after exposure, and cells were stained with 5% Giemsa. A total of about 2000 cells were observed from each group and the mitotic index was calculated. Structural aberrations, including both chromosome and chromatid (i.e., break, deletion, fragments, and exchanges), were recorded. Gaps were recorded but not included in the aberration frequency.

For assessing the acute toxicity study, the test substance was dissolved in distilled water and administered once intramuscularly (intramuscular route is intended for humans) to a single group of five male and five female fasted 10-week-old Sprague-Dawley rats at a dose level of 2000 mg/kg of body weight ( OECD guidelines for testing of chemicals 1987). A concurrent control group was maintained and was treated with the vehicle alone. Animals were received from Animal House, Indian Institute of Toxicology, Pune. All the animals were observed for 14 days for mortality. Clinical observations and body weight were recorded daily. Gross necropsy was performed on all live animals at the end of the study period.

RESULTS AND DISCUSSION

The results of the Ames mutagenic assays indicated that the mean number of histidine revertants in the treatment groups, with and without metabolic activation of the respective strains, were comparable to the mean number of revertants in the solvent control group.

The parallel positive control groups induced 4.72 to 23.36 fold revertants without metabolic activation and 3.00 to 111.61 fold revertants with metabolic activation in assay 1, and 5.18 to 24.75 fold revertants without metabolic activation and 4.16 to 98.32 fold revertants in assay 2 when compared to the respective solvent control group (Table 1).

The mitotic indices for the in vitro chromosomal aberration assay at 45 mg of test substance were 1.93 and 2.31 compared to 2.56 and 2.69 in the control group without and with metabolic activation, respectively. The mitotic activity was reduced to 14.13% and 24.61% with and without metabolic activation. The mitotic activity at 22.5 and 11.25 mg per culture was found to be comparable with the vehicle-control group with and without metabolic activation (Table 2).

With metabolic activation, the number of chromosomal aberrations per cell was 0.015, 0.00, and 0.00 at 45 mg, 22.5 mg, and 11.25 mg per culture; 0.00 in the vehicle-control group; and 0.10 in the positive-control group. The aberrations induced were of chromatid type only. Without metabolic activation, the number of chromosomal aberrations per cell was 0.013, 0.011, and 0.011 at 45 mg, 22.5 mg and 11.25 mg per culture, respectively; 0.012 in the vehicle-control group; and 0.12 in the positive-control group (Table 2). The aberrations induced were of chromatid type only.

The results of the mutagenesis assay indicate that the test substance is nonmutagenic to any test strains used in the presence and absence of S-9 fraction, indicating that there is remote possibility of generation of any mutagenic derivatives in vivo during metabolism.

The results of the in vitro chromosomal assay indicate that the test substance did not induce chromosomal aberrations in cultured mammalian cells up to the maximum recommended concentration of 45 mg per culture and is nonclastogenic in mammalian cells in vitro. Similar observations have been reported for cefotaxime ( AHFS drug information 2002; Aventis Pharmaceuticals 2002).

Based on the results of these studies, it is concluded that the test substance is nonclastogenic and nonmutagenic in the presence and absence of metabolic activation at the maximum recommended concentration of 45 mg per culture for the in vitro chromosomal aberration assay and 50 μg/plate for the mutagenicity assay.

Results of the acute intramuscular toxicity study in rats demonstrated that the test substance is found to be well tolerated in Sprague-Dawley rats. All the animals survived throughout the study period. No clinical signs of intoxication were observed in animals treated at the dose level of 2000 mg/kg body weight. Body weight gain of male and female treated animals was found to be comparable with control on days 7 and 14 (Figure 2). Macroscopic observations of the animals revealed no appreciable difference in pathological findings between the treated and control groups. The median lethal dose (LD₅₀) of the test sub-stance was found to be greater than 2000 mg/kg body weight, which is similar to cefotaxime in rats (NKRZAZ Chemotherapy 1980).