Mutagenicity (Ames test)

Mutagenic activity of IDM01 was performed in full compliance with the OECD guidelines for mutagenicity testing Test No: 471[35,36] and USFDA (40 CFR 79.68). The bacteria used were histidine auxotrophic strains of Salmonella typhimurium (TA97a, TA 98, TA 100, TA 1535 and TA 102). All the strains were stored in liquid nitrogen (−160°C), in cryocans. Strains were maintained on master plates and checked periodically for viable counts and genotype characteristics. For the assay, cultures were grown for 16 h in nutrient broth at 37°C. The cell density of cultures, 1 × 10⁹ cells per ml, was assessed by cell count.

IDM01 was dissolved in sterile water for injection and was tested in plate incorporation assay at a concentration of 5000.00, 1666.67, 555.55, 185.18, and 61.72 μg/plate. The concentrations selected were based on dose range finding study that used concentration range of 5000–1 μg/plate; there was no cytotoxicity observed up to the concentration of 5000 μg/plate. The number of groups formed was 7 and were kept for incubation for 48–72 h at 37°C. Minimal glucose agar medium (Vogel-Bonner minimal medium E), soft agar (overlay agar containing 0.5 mM histidine and biotin, Minimal glucose agar with biotin, Minimal glucose agar with histidine and biotin), minimal glucose agar (histidine, biotin, and ampicillin), nutrient agar, and nutrient broth mediums were prepared. The bacterial strains were cultured in nutrient broth. Vogel-Bonner minimal medium E was chosen as the selective medium. The top agar contained 0.6% agar, 0.5% NaCl, and 0.05 mM histidine-biotin.

Sprague-Dawley male rats (6–8 weeks old, 175–200 g), bred at the Indian Institute of Toxicology (Pune, Maharashtra, India), were used. They were used for the preparation of liver homogenate S-9 mix fraction. Mixed function oxidase systems in the rat liver were stimulated following an intraperitoneal injection of sodium phenobarbital (diluted in corn oil) at a dosage of 80 mg/kg/day for 5 consecutive days. On the 6^th day of induction, following an overnight fasting, the rats were killed and livers aseptically removed.[37]

The preparation of liver homogenate was carried out with sterile glassware and solutions at 4°C. Excised samples of liver were transferred to a beaker containing 0.15 M KCl. After weighing and washing, livers were transferred to a beaker containing 0.15 M KCl (3 ml KCl: 1 g liver) minced with sterile scissors and homogenized. The homogenate was centrifuged for 10 min at 9000 r/min and the supernatant divided into small aliquots. These were stored at − 160°C in cryocan and tested with mutagen 2-aminofluorene (2-AF) before use.

Metabolic activation was performed using a cofactor supplemented postmitochondrial fraction (S9 fraction). The characterization of S-9 preparation was carried out by total protein estimation (3.83 g/100 ml, Lowry method) and sterility of S-9 preparation by examining the plates that had no microbial contamination. The S-9 mix was prepared immediately before its use in the experimental procedure. The microsomal enzyme reaction mixture contained S-9 (1.00 ml/10 ml), 0.4 M MgCl2, 1.65 M KCl salt solution (0.20 ml/10 ml), 1.0 M G-6-P (0.05 ml/10 ml), 0.1 M NADP (0.40/10 ml), 0.2 M Phosphate buffer pH 7.4 (5.00 ml/10 ml), and distilled water (3.35/10 ml).

The strain-specific positive control chemicals used in nonactivation studies are 4-Nitroquinolene-N-Oxide at 0.5 μg/plate for TA 98/TA 97a strains; methyl methanesulfonate at 1 μl for TA 100 and TA 102 strain, and sodium azide at 0.5 μg/plate for TA 1535 strain. The positive control chemicals used in the presence of metabolic activation are 2-AF at 10 μg/plate for TA 98, TA 97a and TA 100 whereas 2-aminoanthracene at 0.5 μg/plate and danthron at 30 μg/plate were used as positive control for TA 1535 and TA 102 strain. VC and phosphate buffer was used as negative control for nonactivated and activated procedure, respectively. Overnight cultures were prepared by transferring a colony from the appropriate master plate to 25 ml of nutrient broth. Inoculated cultures were kept incubated for 16 h at 37°C temperature to achieve a cell density of about 10⁹ cells/ml.

For experiment without metabolic activation, a cell suspension of a tester strain (0.1 ml), 0.5 ml of phosphate buffer, and 100 μl of the vehicle or freshly prepared test substance were added to 2 ml of top agar with L-histidine-biotin. These mixtures were overlaid on the hardened surface of 25 ml minimal glucose agar plates. The plates were inverted and incubated at 37°C for 48 h. Each concentration was tested in triplicates. For the experiment with metabolic activation, above-mentioned procedures were repeated except that 0.5 ml of the S-9 mix was added to the top agar instead of 0.5 ml of phosphate buffer.

The plates were checked for sterility and scored for a uniform lawn of auxotrophs (his-) and the colonies of histidine revertants as the prototrophs. Histidine revertant colonies were counted manually. The mean number of histidine revertants for all the treatment groups was compared with the number of revertants in the respective VC group. The mutagenic activity of the test substance was considered for positive in case of increased concentration over the range tested and a reproducible increase at one or more concentrations in a number of revertant colonies per plate in at least one strain with or without metabolic activation system. The test substance was considered to be toxic if there was a decrease in the number of revertants and/or thinning or absence of background lawn.