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

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
An amount of 0.0103 g of test item was weighed into a 5 mL volumetric flask and 2 mL of acetonitrile was added to it. 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.
- Sampling intervals/times for sterility check: 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.

Buffers:

- pH: 4.0, 7.0 and 7.0
- Type and final molarity of buffer: 0.2 M acetic acid solution, 0.2 M of monobasic sodium phosphate solution and 0.1 M of boric acid solution was used during the study.
- Composition of buffer:
Buffer Solution of pH 4.0:
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.

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.

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.

- Other: All buffer solutions were sterilized by passing through 0.2 μm sterilized filters.

Estimation method (if used):

Not applicable

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 20 ml amber-coloured glass containers. Test solution for each pH along with respective control samples were contained in amber-colored glass vessels covered with aluminium foil and parafilm and capped with PTFE caps and glass stoppers.
- Sterilisation method: Empty vessels and stoppers were sterilized by autoclaving at 121ºC for 20 mins prior to use.
- Measures taken to avoid photolytic effects: yes, prepared test chemical solutions were taken in amber-colored volumetric flasks and kept in a dark incubatoer in order to avoid photolytic effects.
- Measures to exclude oxygen: the buffer solutions were bubbled with nitrogen to avoid oxygen.
- If no traps were used, is the test system closed/open: yes, test system is closed with PTFE caps and glass stoppers.
- Is there any indication of the test material adsorbing to the walls of the test apparatus?: no

TEST MEDIUM
- Volume used/treatment
- Kind and purity of water:
- Preparation of test medium:
- Renewal of test solution:
- Identity and concentration of co-solvent:

Duration:

5 d

Temp.:

50 °C

Initial conc. measured:

15 mg/L

Remarks:

Study was performed at pH 4.0, 7.0 and 9.0, respectively.

Number of replicates:

3 replicates

Positive controls:

not specified

Negative controls:

not specified

Transformation products:

not measured

Key result

Temp.:

25 °C

DT50:

> 1 yr

Remarks on result:

other: Tier 1 approach, Study was performed at pH 4.0, 7.0 and 9.0 and at a temp. of 25°C, respectively.

Remarks:

(Tier I approach)

Details on results:

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), it is presumably stable in water.

Hydrolysis Preliminary Test (Tier-I)

The test samples in pH 4.0, 7.0, and 9.0 buffer solutions were subjected to hydrolysis at 50 + 0.5°C. Triplicate samples were analyzed along with respective control samples for analyte concentration on day 0 and 5 days of post treatment.

Representative chromatograms are presented from Figure 15 to 20.

Hydrolysis at pH 4.0

The mean analyte concentration in the sterile pH 4.0 buffer samples were 14.86 pg/mL and 14.32 g/mL at 0 and 5 days of post treatment, respectively.

The hydrolysis (%) of the analyte in the sterile pH 4.0 buffer samples was 3.63% after 5 days of post treatment. The results are presented in Table 7.

Hydrolysis at pH 7.0

The mean analyte concentration in the sterile pH 7.0 buffer samples were 15.06 g/mL and 14.94 pg/mL at 0 and 5 days of post treatment, respectively.

The hydrolysis (%) of the analyte in the sterile pH 7.0 buffer samples was 0.80% after 5 days of post treatment. The results are presented in Table 7.

Hydrolysis at pH 9.0

The mean analyte concentration in the sterile pH 9.0 buffer samples were 15.18 pg/mL and 14.52 pg/mL at 0 and 5 days of post treatment, respectively.

The hydrolysis (%) of the analyte in the sterile pH 9.0 buffer samples was 4.35% after 5 days of post treatment. The results are presented in Table 7.

The preliminary test results revealed that the hydrolysis of analyte after 5 days of incubation at 50 + 0.5°C is detailed in below table for different buffer solutions.

Time interval	Test chemical (mg/l)			%Hydrolysed
	pH 4.0	pH 7.0	pH 9.0	pH 4.0	pH 7.0	pH 9.0
Initial	14.86	15.06	15.18	-	-	-
Day 5	14.32	14.94	14.52	3.63	0.80	4.35

Detector Linearity (DLC)

A series of working standard solutions of concentration 0.50 mg/L to 25.01 mg/L for Solvent Blue 122 were injected in triplicate to HPLC. The detector response was found linear in this concentration range with a correlation coefficient (r) of 0.9993.

For details refer Table 1 to 3 and Figure 1 to 3. Representative chromatogram is presented in Figure 4.

Limit of Detection (LOD)

The minimum quantity of the standard of 0.5 mg/L, was detected by the HPLC with one-third of LOQ concentration.

Limit of Quantification (LOQ)

The lowest concentration of the reference standard of 1.5 mg/L (DLC-1) analyzed using the method being validated. The accuracy and precision at this

concentration was found to be acceptable. The results are presented in Table 4 to 6.

Validity criteria fulfilled:

yes

Conclusions:

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.

Executive summary:

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.

Description of key information

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.

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

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.