N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

Endpoint summary

Description of key information

Hydrolysis

Hydrolysis study of test chemical was carried out at pH 4.0, 7.0 and 9.0 for determining the half-life value of test chemical. Study was performed in accordance with the OECD Guideline 111 (Hydrolysis as a Function of pH).Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. High Performance Liquid Chromatograph (HPLC) was equipped with DAD and PC based data system. Column used was column: Inertsil ODS-3V, [250 mm x 4.6 mmi.d. x 5 μm particle size] or equivalent. Mobile phase used in the study was Milli-Q water and Acetonitrile, respectively. It has a detector wavelength of 210 nm, flow rate of 1.0 ml/min, injection volume of 100 μl. All the parameters were maintained constant throughout the analysis. The analyte peak in the sample was identified by comparing its retention time with that of analyte peak in reference standard (absence of such a peak in control was also checked). Calibration curve was prepared for the analyte by plotting peak area versus concentration (μg /mL), corrected for purity, for each standard. Best-line fit equation (Y = a + bX) was calculated using the method of least squares. In this equation, ‘Y’ is peak area, ‘X’ is concentration (μg/mL), ‘a’ is Y-intercept and ‘b’ is the slope of the line. For LOD, the minimum quantity of the analyte, which was detected by the HPLC with one-third of LOQ concentration, was determined. An aliquot of 0.2 mL of the DLC-7 solution was diluted to 10 mL with acetonitrile and the same solution was injected as DLC-1 to check the LOD of the method. The LOQ of the equipment for the analyte was determined by analyzing lowest concentration of the reference standard solution (1.5 mg/L) with an acceptable accuracy and precision. For the linearity range, a stock solution of 2033.22 mg/L was prepared by dissolving accurately weighed quantity of 0.0103 g of test chemical reference standard (98.7%) in 5 mL volumetric flask. The contents were dissolved by adding about 2.0 mL of acetonitrile by sonication for 5 minutes. After equilibrating to room temperature, the volume was made up with acetonitrile.Later, the solutions for detector linearity were prepared by diluting known aliquots of the stock solution to a known volume with acetonitrile. Each of the standard solutions from DLC 1 to DLC 7 was injected in triplicate and the detector response (peak area) for each injection was recorded. A graph of the peak area (Y-axis) versus concentration (X-axis) was plotted and the intercept (a), slope (b) and linear regression coefficient (r) were calculated. A test chemical solution was prepared by dissolving 0.0103 g of test chemical in 2 ml of acetonitrile into a 5 ml of volumetric flask. The contents were sonicated for 2 minutes and finally the volume was made up to the mark with acetonitrile. The concentration of the stock solution was 2033.22 mg/L (calculated considering the purity of the test item as 98.7%). An aliquot of 0.075 mL of the same was fortified in pH 4, 7 and 9 buffer solutions at the concentration of 15.25 mg/L (with <1% co-solvent) in duplicate and incubated for 5 days at 50 + 0.5°C along with untreated buffer solutions. The test samples were prepared in laminar flow chamber under aseptic comditions. Sterility of the samples were checked at both start and end of the hydrolysis experiment for each of the test system. 0.2 M acetic acid solution, 0.2 M of monobasic sodium phosphate solution and 0.1 M of boric acid solution were used as a buffer solution during the study. For preparing the buffer Solution of pH 4; an aliquot of 205 mL of 0.2M acetic acid solution and 45 mL of 0.2M anhydrous sodium acetate solution was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 4.01.for preparing the buffer solution of pH 7.0; an aliquot of 97.5 mL of 0.2M monobasic sodium phosphate solution and 152.5 mL of 0.2 M dibasic sodium phosphate solution were transferred into a 1000 mL volumetric flask. The contents were diluted to volume with Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 7.01 and for preparing the buffer solution of pH 9.0; an aliquot of 500 mL of 0.1M boric acid solution and 500 mL of Milli-Q® water was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 9.01. All buffer solutions were sterilized by passing through 0.2 μm sterilized filters. 20 ml amber-coloured glass containers was used as a test vessel. Prepared test chemical solutions were taken in amber-colored volumetric flasks and kept in a dark incubatoer in order to avoid photolytic effects. The buffer solutions were bubbled with nitrogen to avoid oxygen.Test system was kept closed with PTFE caps and glass stoppers. Empty vessels and stoppers were sterilized by autoclaving at 121ºC for 20 mins prior to use. Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. All experiments were performed in triplicates. The mean analyte concentration in the sterile pH 4.0, 7.0 and 9.0 buffer samples were 14.86, 15.06, 15.18 pg/mL and 14.32, 14.94 and 14.52 g/mL at 0 and 5 days of post treatment, respectively. Percentage degradation of test chemical was determined to be 3.63%, 0.80% and 4.35% at pH 4.0, 7.0 and 9.0 at a temperature of 50°C, respectively. The half-life value of test chemical was determined to be > 1 year. Since the test chemical is not degraded more than 10% in the test conditions (Tier-1), test chemical was considered to be stable in water.

Additional information

Hydrolysis

Hydrolysis study of test chemical was carried out at pH 4.0, 7.0 and 9.0 for determining the half-life value of test chemical. Study was performed in accordance with the OECD Guideline 111 (Hydrolysis as a Function of pH).Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. High Performance Liquid Chromatograph (HPLC) was equipped with DAD and PC based data system. Column used was column: Inertsil ODS-3V, [250 mm x 4.6 mmi.d. x 5 μm particle size] or equivalent. Mobile phase used in the study was Milli-Q water and Acetonitrile, respectively. It has a detector wavelength of 210 nm, flow rate of 1.0 ml/min, injection volume of 100 μl. All the parameters were maintained constant throughout the analysis. The analyte peak in the sample was identified by comparing its retention time with that of analyte peak in reference standard (absence of such a peak in control was also checked). Calibration curve was prepared for the analyte by plotting peak area versus concentration (μg /mL), corrected for purity, for each standard. Best-line fit equation (Y = a + bX) was calculated using the method of least squares. In this equation, ‘Y’ is peak area, ‘X’ is concentration (μg/mL), ‘a’ is Y-intercept and ‘b’ is the slope of the line. For LOD, the minimum quantity of the analyte, which was detected by the HPLC with one-third of LOQ concentration, was determined. An aliquot of 0.2 mL of the DLC-7 solution was diluted to 10 mL with acetonitrile and the same solution was injected as DLC-1 to check the LOD of the method. The LOQ of the equipment for the analyte was determined by analyzing lowest concentration of the reference standard solution (1.5 mg/L) with an acceptable accuracy and precision. For the linearity range, a stock solution of 2033.22 mg/L was prepared by dissolving accurately weighed quantity of 0.0103 g of test chemical reference standard (98.7%) in 5 mL volumetric flask. The contents were dissolved by adding about 2.0 mL of acetonitrile by sonication for 5 minutes. After equilibrating to room temperature, the volume was made up with acetonitrile.Later, the solutions for detector linearity were prepared by diluting known aliquots of the stock solution to a known volume with acetonitrile. Each of the standard solutions from DLC 1 to DLC 7 was injected in triplicate and the detector response (peak area) for each injection was recorded. A graph of the peak area (Y-axis) versus concentration (X-axis) was plotted and the intercept (a), slope (b) and linear regression coefficient (r) were calculated. A test chemical solution was prepared by dissolving 0.0103 g of test chemical in 2 ml of acetonitrile into a 5 ml of volumetric flask. The contents were sonicated for 2 minutes and finally the volume was made up to the mark with acetonitrile. The concentration of the stock solution was 2033.22 mg/L (calculated considering the purity of the test item as 98.7%). An aliquot of 0.075 mL of the same was fortified in pH 4, 7 and 9 buffer solutions at the concentration of 15.25 mg/L (with <1% co-solvent) in duplicate and incubated for 5 days at 50 + 0.5°C along with untreated buffer solutions. The test samples were prepared in laminar flow chamber under aseptic comditions. Sterility of the samples were checked at both start and end of the hydrolysis experiment for each of the test system. 0.2 M acetic acid solution, 0.2 M of monobasic sodium phosphate solution and 0.1 M of boric acid solution were used as a buffer solution during the study. For preparing the buffer Solution of pH 4; an aliquot of 205 mL of 0.2M acetic acid solution and 45 mL of 0.2M anhydrous sodium acetate solution was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 4.01.for preparing the buffer solution of pH 7.0; an aliquot of 97.5 mL of 0.2M monobasic sodium phosphate solution and 152.5 mL of 0.2 M dibasic sodium phosphate solution were transferred into a 1000 mL volumetric flask. The contents were diluted to volume with Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 7.01 and for preparing the buffer solution of pH 9.0; an aliquot of 500 mL of 0.1M boric acid solution and 500 mL of Milli-Q® water was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 9.01. All buffer solutions were sterilized by passing through 0.2 μm sterilized filters. 20 ml amber-coloured glass containers was used as a test vessel. Prepared test chemical solutions were taken in amber-colored volumetric flasks and kept in a dark incubatoer in order to avoid photolytic effects. The buffer solutions were bubbled with nitrogen to avoid oxygen.Test system was kept closed with PTFE caps and glass stoppers. Empty vessels and stoppers were sterilized by autoclaving at 121ºC for 20 mins prior to use. Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. All experiments were performed in triplicates. The mean analyte concentration in the sterile pH 4.0, 7.0 and 9.0 buffer samples were 14.86, 15.06, 15.18 pg/mL and 14.32, 14.94 and 14.52 g/mL at 0 and 5 days of post treatment, respectively. Percentage degradation of test chemical was determined to be 3.63%, 0.80% and 4.35% at pH 4.0, 7.0 and 9.0 at a temperature of 50°C, respectively. The half-life value of test chemical was determined to be > 1 year. Since the test chemical is not degraded more than 10% in the test conditions (Tier-1), test chemical was considered to be stable in water.