Received: 24 Aug 2011, Revised and Accepted: 16 Nov 2011 ABSTRACT Ames bacterial reverse mutation assay is one of the basic screening tools to evaluate the mutagenicity of chemicals. The solubility of test compounds plays a major role in deciding the highest concentration for the Ames assay. The selection of a solvent for new chemical entities, which is compatible with Salmonella typhimurium, poses a challenging task for genetic toxicologists in pharma industries. We have selected Dimethyl sulfoxide, absolute ethanol, acetone and acetonitrile to evaluate its compatibility with Salmonella as compared to water using plate incorporation method. Observations in the present study revealed that all these solvents were found to be compatible as indicated by no significant changes in the bacterial revertatnt colony counts and hence can be continued as vehicles in Ames bacterial reverse mutation assay. Research Article COMPATIBILITY OF DIFFERENT SOLVENTS WITH SALMONELLA TYPHIMURIUM MUTANT STRAINS IN BACTERIAL REVERSE MUTATION ASSAY RAVICHANDRA BHADRAVATHI VEDMAURTHY 1* , SRIRAM PADMANABHAN 1 , MAGESH VIJAYAN 2 , ZABIULLAH ALANDUR JAMAL 2 , JACOB KUNJUMMAN 2 , MATHURAM LALGUDI NARAYANAN 1 1 Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Vepery Chennai- 600 007, India, 2 Department of Toxicology, Orchid Research Laboratories Ltd., Sholinganallur, Chennai 600019, India. *Email: rachan673@gmail.com Keywords: Ames assay, Compatibility, Salmonella typhimurium, Solvent INTRODUCTION The Salmonella Ames assay is one of the most widely used short- term basic regulatory tests to assess the mutagenic potential of new chemical entities 1 . The solubility of the chemicals plays a very pivotal role in deciding the concentration for the Ames mutagenicity assay. Poorly soluble or water insoluble drugs are often a challenging task for formulators in the pharma industry. Apart from universal solvent (water), there are many organic solvents like DMSO, ethanol, acetone acetonitrile, etc which can be used as a vehicle in Ames assay 1 . Very few literatures depict the compatibility of the various solvents in Ames salmonella reverse mutation assay. By considering the lack of literature, the present experiment was planned to assess the compatibility of different solvents (DMSO, absolute ethanol, acetone and acetonitrile) with salmonella mutant strains. MATERIALS AND METHODS The Salmonella typhimurium tester strains TA97a and TA98 used in the present study were procured from Bruce Ames laboratory, USA. All the chemicals, reagents and positive controls used in the present study were obtained from sigma Aldrich, USA. Rat liver S9 fraction was prepared in house from male Sprague Dawley rats. The method used in present study followed as per the recommendations of Maron and Ames (1983) 2 and OECD guideline 471 (1997) 3 . Briefly, bacterial culture (100µL) was exposed to different solvents (100µL; Water, absolute ethanol, DMSO, acetone and acetonitrile) both in presence (500 µL 5 % S9) and absence (500 µL of phosphate buffer) of metabolic activation systems by plate incorporation method. The plates were incubated at 37 0 C for 72 hours and revertant colonies were counted manually. The data was evaluated and interpreted biologically and no statistical analysis was performed. RESULTS & DISCUSSION In general, in neither of the test compounds, no significant changes (increase or decrease) in the mean revertant colony counts were observed in both the tester strains (TA97a and TA98) as compared to negative control (water) in presence and absence of metabolic activation system. The revertant colony counts for different solvents are shown in the table 1. Table 1: Bacterial reverse mutation assay using Salmonella typhimurium: his + revertant colony counts Without metabolic activation system Strain / Revertant colony counts per plate Water DMSO Absolute ethanol Acetone Acetonitrile TA97a Individual Plate Count (n=3) 136 129 135 128 116 147 125 149 125 128 123 132 158 122 141 Mean ± SD 133.33 ± 3.79 130.33 ± 15.63 133.00 ± 13.86 127.67 ± 4.51 140.33 ± 18.01 TA98 Individual Plate Count (n=3) 17 17 21 17 25 18 23 12 19 29 21 23 25 27 21 Mean ± SD 18.33 ± 2.31 20.00 ± 4.36 18.00 ± 5.57 24.33 ± 4.16 24.33 ± 3.06 With metabolic activation system (5 % S9 v/v) Strain / Revertant colony counts per plate Water DMSO Absolute ethanol Acetone Acetonitrile TA97a Individual Plate Count (n=3) 135 130 141 135 130 129 140 135 132 140 106 136 141 138 130 Mean ± SD 135.33 ± 5.51 131.33 ± 3.21 135.67 ± 4.04 127.33 ± 18.58 136.33 ± 5.69 TA98 Individual Plate Count (n=3) 31 28 26 31 24 28 26 24 26 29 24 26 25 27 25 Mean ± SD 28.33 ± 2.52 27.67 ± 3.51 25.33 ± 1.15 26.33 ± 2.52 25.67 ± 1.15 In the present study, ethanol was found to be non-mutagenic, non- cytotoxic and proved to be a compatible solvent with Salmonella bacteria. Our findings were found similar to that obtained by other authors 4,5 . Oxidative metabolite (acetaldehyde ) of ethanol is a proven mutagenic and carcinogenic in mammalian cells, but not in bacterial system as evinced by negative mutagenicity results in Salmonella and E.coli bacterial reverse mutation assays 6 and our findings were also in concordance with their findings. In mammalian cells, acetaldehyde induces genotoxicity by DNA-DNA and DNA- protein cross linking mechanism. In the present study, the absence of mutagenicity of ethanol even in the presence of metabolic activation system containing alcohol dehydrogenase essential for oxidation of ethanol might be attributed to the lack of formation of DNA cross linking unlike in mammalian cells. DMSO being used as most common solvent after water in many laboratories since long time 7 . According to earlier published reports, DMSO could be used as alternate solvent when chemicals do not dissolve in water 1 . But DMSO had induced mutagenicity in TA1537 and TA2637 strains of Salmonella typhimurium both in the absence International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 4, Issue 1, 2012 A A c c a a d d e e m mi i c c S Sc c i i e e n nc c e e s s