Synacinn™: Bacterial reverse mutation test data in five histidine-requiring strains of Salmonella Typhimurium

The present data described the analysis of mutagenicity in SynacinnTM by assessing the point mutations occurring due to Synacinn™ exposure to five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102), in the presence or absence of an exogenous mammalian metabolic activation system (S9). It was conducted in two Phases - Phase I (Dose Range Finding experiment-DRF) and Phase II (Mutagenicity Assay 1 and 2). DRF and Mutagenicity Assay 1 was conducted employing plate incorporation method, while Mutagenicity Assay 2 was performed using pre-incubation method. Formulation analysis pertaining to SynacinnTM was performed for both Mutagenicity Assay 1 and 2. Dose formulations were prepared fresh on each day of the experiment. Adventol 50% v/v in purified water was selected as a suitable vehicle based on the preliminary solubility test. Based on the Phase I analysis, 5 mg/plate was selected as the highest concentration of SynacinnTM followed by lower concentrations of 2.5, 1.25, 0.625 and 0.313 mg/plate for the Mutagenicity Assays. Genetic integrity of all the tester strains used was confirmed by performing genotyping before their use. All the data acceptability criteria were fulfilled confirming the validity of the test.


a b s t r a c t
The present data described the analysis of mutagenicity in Synacinn TM by assessing the point mutations occurring due to Synacinn TM exposure to five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102), in the presence or absence of an exogenous mammalian metabolic activation system (S9). It was conducted in two Phases -Phase I (Dose Range Finding experiment-DRF) and Phase II (Mutagenicity Assay 1 and 2). DRF and Mutagenicity Assay 1 was conducted employing plate incorporation method, while Mutagenicity Assay 2 was performed using pre-incubation method. Formulation analysis pertaining to Synacinn TM was performed for both Mutagenicity Assay 1 and 2. Dose formulations were prepared fresh on each day of the experiment. Adventol 50% v/v in purified water was selected as a suitable vehicle based on the preliminary solubility test. Based on the Phase I analysis, 5 mg/plate was selected as the highest concentration of Synacinn TM followed by lower concentrations of 2.5, 1.25, 0.625 and 0.313 mg/plate for the Mutagenicity Assays. Genetic integrity of all the tester strains used was confirmed by performing genotyping before their use. All the data acceptability criteria were fulfilled confirming the validity of the test.  Table   Subject Biological sciences Specific subject area Microbiology: Applied Microbiology Type of data Table  How data were acquired Revertant colonies grown on Minimal Agar plates were counted manually. Background bacterial lawn was observed using microscope and scored. Data format Raw Analyzed Parameters for data collection After incubation for 48-72 hours (DRF and Mutagenicity Assay 1 and 2), plates were removed from the incubator and each treatment plate was assessed for precipitation and cytotoxicity. Description of data collection Precipitation was graded based on the amount of visible precipitate on the plate. Cytotoxicity was assessed in terms of diminution of background bacterial lawn, presence of micro colonies and/or reduction in the number of revertant colonies. The fold increase in revertant colony counts for each test item treatment versus the vehicle control group was determined to assess the mutagenic potential of Synacinn TM

Value of the Data
• These data provide information on the mutagenicity potential of SynacinnTM using five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102) in the absence and presence of an exogenous metabolic activation system (S9). • These data are useful for researchers who want to determine the mutagenicity potential of their polyherbal formulations. • SynacinnTM is currently approved by National Pharmaceutical Regulatory Agency (NPRA), Malaysia as traditional medicine with a general health claim. Data on the mutagenicity potential of SynacinnTM are valuable to establish the safety pharmacology for SynacinnTM and can be used in future for the development process and approval of SynacinnTM as a prescription botanical medicine for diabetes.

Data Description
All markers identified in Synacinn TM formulations (Mutagenicity assay 1 and 2: raw data as in Supplementary Table 1S) was presented in Table 1 for all dose strengths (6.25 mg/mL, 12.5 mg/mL, 25 mg/mL and 50 mg/mL). Table 2 shows the peak area of catechin from HPLC analysis of top, middle and bottom layers of Synacinn TM formulations in triplicate. In the Dose Range

Formulation analysis
Synacinn TM powder was provided by Proliv Life Sciences Sdn Bhd and standardized with five selected markers as follows: rosmarinic acid -0.41% w/w, andrographolide -0.25% w/w, catechin -1.48% w/w, curcumin -0.11% w/w and gallic acid -1.56% w/w. Andrographolide (sc-205594A), catechin (sc-204673A), curcumin (sc-200509A) and gallic acid (sc-205704A) were purchased from Santa Cruz Biotechnology, while rosmarinic acid (sc-202796A) was purchased from Chengdu Biopurify Phyto Chemicals Ltd. All test items were stored at room temperature protected from light. Identification of Synacinn TM markers was done for the formulation analysis by comparing the retention time of each marker in all the dose strengths against the identification solution. Only one marker (catechin) was quantified by reporting the peak area and Percentage Relative Standard Deviation (%RSD = Standard Deviation/ Mean x 100; acceptance limit of %RSD ≤ 20%) to evaluate homogeneity of the formulations [1] . All formulations (6.25, 12.5, 25, 50 mg/mL) were pipetted out from top, middle and bottom layer in triplicates, injected once into HPLC and chromatograms were recorded.

Marker stock solution preparation
Each marker was weighed (2 mg) transferred into five separate 10 mL volumetric flask. 5 mL of methanol was added to each flask to dissolve completely and made up the volume to 10 mL with water and mix well.

Marker solution preparation (0.01 mg/mL)
Each of the marker stock solution was pipetted out (1.0 mL) and transferred into 20 mL volumetric flask. The volume was made up to 20 mL with diluent and mixed well.

Identification solution preparation
Synacinn was weighed (50 mg) and transferred into 10 mL volumetric flask. 5 mL of diluent was added and each of the marker stock solution was spiked (0.5 mL) into 10 mL volumetric flask. The volume was made up to 10 mL with diluent and mixed well. The solution was

Test System
Salmonella typhimurium strains of TA1537, TA1535, TA98, TA100 and TA102 were used in this experiment as per the test guidelines OECD 471 and ICH S2 (R1) [2,3] . Each tester strain was characterized and confirmed on their genotypes. The integrity of the tester strains was tested by verifying its histidine requirement, sensitivity to UV radiation, resistance to ampicillin/ tetracycline and rfa mutation. Only qualified batches of strains were employed in the experiments. All tester strains were maintained as frozen permanent stocks (Cryovial working stocks) and stored in ultra-deep freezer at approximately -70 °C. Frozen aliquots of bacterial culture were thawed and a fixed inoculum was added to a flask containing Oxoid Nutrient Broth (ONB-2). Inoculated flask was incubated overnight (15-16 h) in an incubator equipped with a shaker at 37 °C with shaking [110 revolutions per minute (rpm)]. These overnight grown cultures containing 10 9 CFU/mL were used for the assay conduct. Optical density was determined using spectrophotometer at 650 nm. Actual cell titers were determined by viable count on nutrient agar plates for each assay and recorded as raw data.

Vehicle selection
Preliminary test was conducted to select appropriate vehicle for the experiment and to assess test item solubility. For vehicle selection, small amount of test item was taken to achieve 50 mg/mL concentration. Test item solubility was checked in different vehicles in preferential order starting with water, DMSO and 50% adventol. Test item did not form clear solution in any of the tested solvents. The formulations were vortexed for 5-10 minutes and centrifuged at 10 0 0 rpm for 5 minutes to remove debris or non-active polysaccharides from purified formulation (supernatant) containing selected makers [4] . The supernatant from each formulation was carefully collected and submitted for analytical method feasibility. Formulation prepared in adventol 50% was most suitable for identification of all the five markers. Therefore, based on the analytical method feasibility, adventol 50 % v/v in purified water was preferred over water and DMSO and selected vehicle for this experiment. Moreover, adventol (99% ethanol) is one of the recommended solvents, biocompatible to Salmonella tester strains [5,6] . Adventol (manufactured by Advent Chembio Private Limited) was stored at room temperature.

Mammalian Liver Post Mitochondrial Fraction (S9)
The mammalian liver post mitochondrial fraction (S9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA (MolToxTM S9), where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. Batches of MolToxTM S9 were stored frozen at -70 ± 10 °C and thawed just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P 450 catalysed enzyme activities (alkoxyresorufin O dealkylase activities) [7][8][9] .

Preparation of S9 Mix
S9 mix was prepared freshly for each assay by mixing commercially procured S9 fraction with the required cofactors in the ratio of 1:9 which corresponds to 10% v/v of S9 in the final mixture. Cofactor solution were prepared, filtered and aliquoted as per the requirement and stored approximately at -20 °C. Once prepared, S9 mix was maintained on ice throughout its use during the experiment and left over was discarded. The composition of the cofactors used is given in the following Table 14 :

Positive Controls Information
The details of the positive controls used were given in the following Table 15 ;

. Plate incorporation method
In this method, the plating was achieved by the following sequence of additions to 2 mL of molten agar (supplemented with 10% v/v, 0.5 mM Histidine-Biotin solution) maintained at 45 ± 2 °C for treatment without metabolic activation: 0.05/0.1 mL of Synacinn TM or positive or vehicle control solution 0.5 mL of Phosphate buffer solution 0.1 mL of bacterial culture These additions were followed by rapid mixing and pouring on to pre-labelled minimal glucose agar plates. When the agar is set, the plates were inverted and incubated. In case of treatment in presence of metabolic activation, 0.5 mL of S9 cofactor mix was used instead of Phosphate buffer. For untreated (i.e. organism) control, 0.1 mL of respective tester strain was added to 2 mL of top agar followed by rapid mixing and pouring on to pre-labelled minimal glucose agar plates. All the treated plates were incubated at 37 ± 1 °C for 48 to 72 hours and evaluated at the end of incubation.

Pre-incubation method
For Pre-incubation method, a pre-incubation step was included in which, test item solution or control solution, S9 mix or phosphate buffer and bacterial culture were mixed and incubated for 20 minutes at 37 ± 1 °C in a shaking water bath at 75 rpm, then 2 mL of molten agar maintained at 45 ± 2 °C was added to this mixture. The plating and incubation procedure was as described in the routine plate incorporation procedure.

Phase I-Dose Range Finding study (DRF)
This phase was designed to assess the cytotoxicity and precipitation with an objective to select test concentrations for the Phase II-Mutagenicity Assay. DRF was conducted with plate incorporation method using TA100 both with and without metabolic activation (10% S9 cofactor mix) at 8 test concentrations ranging from 0.039 to 5 mg/plate separated by factor of 2. Each control (untreated/vehicle/positive) and test item concentrations will be run in triplicate plates.

Phase II-Mutagenicity assay 1 & 2
The objective of this phase was to evaluate the mutagenic potential of the test item. Mutagenicity assays were carried out using S. typhimurium tester strains TA1535, TA1537, TA98, TA100 and TA102. This experiment was conducted both with and without metabolic activation system (10% S9 cofactor mix) using five test concentrations. Each control (untreated/vehicle/positive) and test item concentrations were run in triplicate plates. Mutagenicity assay 1 and 2 were performed using plate incorporation and pre-incubation methods, respectively. Based on the DRF data, concentrations for the Phase II experiments were selected and provided in the following Table 16 ; Final concentrations represented up to 3 decimals only. In order to obtain all test doses within the validated bracketed range of 5 to 50 mg/mL for dose formulation analysis, test volume was adjusted to 50 μL of 6.25 mg/mL to achieve 0.313 mg/plate.