In vitro genotoxicity of piperacillin impurity-A

The manufacturing and storage of the piperacillin produce different impurities of various concentrations, which may influence the efficacy and safety of the drug. Since no report of genotoxicity data is available on the impurities of piperacillin, further studies were designed and conducted to provide information for establishing the safety profile and qualification of the piperacillin impurity-A. Salmonella typhimurium strains were exposed to Piperacillin impurity-A for Ames tests. Neither increase in number of revertants indicative of mutagenic activity nor inhibition of bacterial growth, indicative of cytotoxicity were observed up to 5 mg/plate both in the presence and absence of metabolic activation. Similarly, chromosomal aberration assay did not reveal any significant alterations up to 5 mg/culture as compared to the negative control both in the presence and absence of metabolic activation (S9 mix). The results of these studies indicate that Piperacillin impurity-A is non-mutagenic in Ames test and non-clastogenic in chromosomal aberration study.


INTRODUCTION
There is a growing concern about the role of impurities in drug substances.Safety regulations require insight into the structure and the amount of impurities present in the drug substance before they can be administered to humans.The presence of small amounts of impurities may influence the efficacy and safety of Piperacillin.The manufacturing and storage process produce impurities at various concentrations and occasionally, the concentration of these impurities crosses the threshold limit and warrants for establishing the safety profile (ICH Guideline, 2000a;Federal Register, 2000b).
Piperacillin an antibiotic of the penicillin group is widely used against a large number of gram-positive and gramnegative microorganisms (AHFS Drug Information, 2002).Various impurities of Piperacillin are reported in PHARMEUROPA (2002).However, no genotoxicity data is available on impurities.Piperacillin impurity-A is a prominent degradation product of Piperacillin that appears *Corresponding author.E-mail: mahesharavind2006@yahoo.co.in.Tel: +91 9840343870.during manufacturing and storage process.As the level of this impurity is comparatively higher and failed to pass the validation criteria of computer-assisted toxicity prediction carried out by "Topkat" software, the present genotoxic studies were designed and conducted to provide the information for establishing the safety profile and qualification of the impurity.

Ames assay
Salmonella typhimurium strains TA97a, TA98, TA100, TA1535 and TA102 were obtained from Bruce Ames Laboratory, Molecular and Cell Biology, University of California, and checked for their viable counts and genotype characteristics.Plate incorporation method (Maron and Ames, 1983) using histidine-dependent strains of S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 in the presence and absence of metabolic activation system (S9 liver fraction) was adopted for assessing the mutagenicity.
Based on the results of cytotoxicity test, piperacllin impurity-A was tested for its mutagenic properties at five different concentrations viz., 5, 2.5, 1.25, and 0.625 mg/plate.100 µl of various concentrations of impurity dissolved in dimethyl sulfoxide were added to 2 ml top agar mixed with 100 µl of bacterial culture and then poured on to a plate containing minimal glucose agar.These plates were incubated at 37 o C for 48 h and his+ revertant colonies were manually counted and the results were shown as the mean of the three plates with standard deviation.The influence of metabolic activation was tested by adding 500 µl of S9 mixture.The experiments were analysed in triplicate and was repeated again to confirm the result.The criteria employed to interpret the results of Ames test as positive were similar to those used in regulatory guidelines (OECD and ICH Guidelines).The number of induced mutation should be at least twice the activity observed in negative control and there must be a reproducible dose response curve.Concurrent positive and negative (dimethyl sulfoxide) controls were used in the study.The standard mutagens used as positive controls in each experiment were without metabolic activation, 4-nitroquinoline 1-Oxide (5 g/plate) for strain TA 97a and TA98, Sodium azide (5 g/plate) for strain TA 100 and TA 1535, Mitomycin-C (0.02 mg/plate) for TA 102.In case of positive controls with metabolic activation, 2-aminoflurene (20 g/plate) for TA97a, TA98, Vijayan et al. 2075 TA100, TA 1535 and TA 102.

Chromosomal aberration assay
Chinese Hamster Ovary cell line obtained from National Centre for Cell Science, Pune were used for in vitro chromosomal aberration study.Monolayer cultures of 80% confluency were cultured at a cell density of 2.3 x 10 5 cells per culture and 24 h after culturing, the cells were exposed to the test substance with and without Aroclor 1254 -induced wistar rats S-9 (Venitt et al., 1990).As no precipitation and reduced mitotic index were recorded for impurity of piperacillin at 5 mg/culture, dose levels of 5, 2.5 and 1.25 mg/culture were selected and exposed to cell cultures in duplicate (U.S. Environmental Protection Agency, 1998).Concurrent positive controls mitomycin-C with out S-9 and benzo(A)pyrene with S-9 and negative control (DMSO) were used for the study.Cell cultures were incubated at 37 o C, harvested at 18 h after exposure and the cells were stained with 5% Giemsa.A total of about 200 metaphases were observed for structural chromosome aberrations, including both chromosomes and chromatids (that is, break, deletion, fragments and exchanges) were recorded.Gaps were recorded but not included in the aberration frequency.

RESULTS AND DISCUSSION
All the strains of S. typhimurium; TA 97a, TA 98, TA 100, TA 102 and TA 1535, exposed to different concentrations of Piperacillin impurity-A, did not show two fold or greater increase in the mean number of revertants as compared to negative control group given in Table 1.All strains used in the study exhibited marked increase (>10 fold) in the number of revertants when treated with positive control agents.The results confirmed the sensitivity of the tester strains to mutagens and thus the validity of the assay.The results indicated that the mean number of histidine revertants in the treatment groups were comparable to the mean number of revertants in the negative control group in all the five S. typhimurium tester strains viz., TA 97a, TA 98, TA 100, TA 102 and TA 1535 both in the absence and presence of metabolic activation.
The impurity of piperacillin up to 5 mg/plate in the presence and absence of metabolic activation was found to be non-mutagenic to all the five S. typhimurium tester strains.
Similarly in vitro chromosomal aberration assay did not reveal any significant alterations up to 5 mg/culture given in Table 2 as compared to the negative control both in the presence and absence of metabolic activation (S9 mix) but the positive controls induced aberration.The chromosomal aberrations recorded per cell in the presence of metabolic activation was 0.01, 0.00 and 0.00 at 5, 2.5 and 1.25 mg per culture respectively and 0.01 in vehicle control and 0.08 in the positive control group.The number of chromosomal aberrations per cell recorded in the absence of metabolic activation was 0.01, 0.00 and 0.00 at 5, 2.5 and 1.25 mg per culture respectively, 0.01 in vehicle control and 0.08 in the positive control group.Piperacillin impurity-A up to 5 mg/ml is found to be nonclastogenic to Chinese Hamster Ovary cell lines both in the presence and absence of metabolic activation.

Table 1 .
Mean plate count of mutagenicity study.