1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 September 2022 to 14 November 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 intervals for the parent/transformation products: Two samples of each pH buffer and one control of each pH buffer were taken immediately after the addition of the test item on day 0 analysis, and on day 3 and day 5 of incubation at 50 ± 0.5ºC during preliminary test.
During Tier 2 test, all the test samples and blank control were analyzed for active content concentration as detailed in the full study report (paragraph 7.6.4) after incubation at 20°C, 35°C, and 50°C.
- Sampling intervals/times for pH measurements: The pH was measured after fortification of the test item stock and at the time of each sampling occasion for each buffer solution and control using a pH meter both for preliminary and Tier 2 tests.
- Sampling intervals/times for sterility check: Sterility was checked at higher kinetic test tier 2 experimental start 0th Hour (before incubation) in all the test systems and at experiment termination as follows: 20th Hour in pH 4, pH 7 and pH 9 buffer solutions incubated at 50°C, at 24th Hour in pH 4, pH 7 and pH 9 buffers at 35°C, at 24th Hour in pH 4 buffer at 20°C and at 96th Hour in pH 7 and pH 9 buffers at 20 °C respectively. The test solutions were prepared aseptically under a biosafety cabinet chamber. A volume of 5 mL of each sample was collected and transferred to the microbiology department for sterility testing.

Buffers:

Buffer solutions at pH 4, 7 and 9 were prepared according to the buffer systems of Clark and Lubs.

Details on test conditions:

TEST SYSTEM
- Sterilisation method: All glassware, Milli-Q water and buffer solutions of pH 4, pH 7 and pH 9 (test system) used in the hydrolysis tests were sterilized using autoclave at 121.0°C for 15 minutes at 15 lbs pressure.
- Lighting: All the hydrolysis tests were carried out in dark conditions.
- Measures taken to avoid photolytic effects: The test tubes were covered with aluminium foil to avoid photolytic effects.
- Measures to exclude oxygen: All necessary measures were taken to avoid the presence of oxygen by bubbling nitrogen for 5 minutes before preparing the solution.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

18.785 mg/L

Remarks:

Tier 1 test.
Mean measured concentration.

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

18.949 mg/L

Remarks:

Tier 1 test.
Mean measured concentration.

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

19.091 mg/L

Remarks:

Tier 1 test.
Mean measured concentration.

Duration:

20 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

18.593 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

20 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

18.8 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

20 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

18.901 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

24 h

pH:

4

Temp.:

35 °C

Initial conc. measured:

18.593 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

24 h

pH:

7

Temp.:

35 °C

Initial conc. measured:

18.758 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

24 h

pH:

9

Temp.:

35 °C

Initial conc. measured:

18.901 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

24 h

pH:

4

Temp.:

20 °C

Initial conc. measured:

18.593 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

96 h

pH:

7

Temp.:

20 °C

Initial conc. measured:

18.758 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Duration:

96 h

pH:

9

Temp.:

20 °C

Initial conc. measured:

18.901 mg/L

Remarks:

Tier 2 test.
Mean measured concentration.

Number of replicates:

2 replicates for each buffer, at each of the temperatures tested

Positive controls:

no

Negative controls:

yes

Remarks:

1 control for each buffer, at each of the temperatures tested

Statistical methods:

Standard calculations according to OECD 111 and pseudo first-order equations

Preliminary study:

The preliminary test results revealed that the rate of hydrolysis (percent degradation) of the test item after 5 days of incubation at 50 ± 0.2°C was 93.47% in pH 4 buffer, 91.95% in pH 7 buffer and 93.50% in pH 9 buffer solutions respectively.

Transformation products:

no

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.071 h-1

DT50:

9.69 h

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.057 h-1

DT50:

12.12 h

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.112 h-1

DT50:

6.17 h

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.167 h-1

DT50:

4.15 h

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

35 °C

Hydrolysis rate constant:

0.114 h-1

DT50:

6.06 h

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.056 h-1

DT50:

12.36 h

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.154 h-1

DT50:

4.49 h

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

35 °C

Hydrolysis rate constant:

0.089 h-1

DT50:

7.81 h

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0.045 h-1

DT50:

15.24 h

Type:

(pseudo-)first order (= half-life)

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.182 h-1

DT50:

3.82 h

Type:

(pseudo-)first order (= half-life)

pH:

4

Temp.:

35 °C

Hydrolysis rate constant:

0.127 h-1

DT50:

5.45 h

Type:

(pseudo-)first order (= half-life)

pH:

4

Temp.:

20 °C

Hydrolysis rate constant:

0.105 h-1

DT50:

6.61 h

Type:

(pseudo-)first order (= half-life)

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

Validity criteria fulfilled:

yes

Conclusions:

The preliminary test was conducted at 50 ± 0.2°C, and after 5 days the test item AP 729 was found to be hydrolytically unstable due to 93.47% degradation in pH 4 buffer, 91.95% degradation in pH 7 buffer and 93.50% degradation in pH 9 buffer test solutions and no formation of breakdown products was observed respectively.
In Tier 2 test, the hydrolysis of the test item was investigated as a function of pH (4, 7 and 9) at three temperatures of 20 ± 0.5°C, 35 ± 0.5°C and 50 ± 0.5°C respectively.
At pH 4, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.1123 hour-1 and 6.17 hours, respectively.
At pH 7, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.0572 hour-1 and 12.12 hours, respectively.
At pH 9, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.0715 hour-1 and 9.69 hours, respectively.
At pH 4, 7 and 9, the total rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.2409 hour-1 and 2.88 hours, respectively.

Executive summary:

The hydrolysis of AP 729 as a function of pH in Sterile Aqueous Buffer Solutions of pH 4, pH 7 and pH 9 was studied according to OECD Guideline 111. The hydrolysis of the test item was determined by analyzing the analyte concentration at the predetermined intervals using a validated HPLC method.
The analysis of duplicate buffer solutions after the addition of the test item indicated that mean recoveries of the test item in pH 4, pH 7 and pH 9 buffer solutions at initial analysis for tier 1 were 91.12%, 91.91% and 92.60% and for tier 2 were 90.35%, 91.14% and 91.84% and thus fell within the quality criteria of 90-110% for hydrolysis experiment. The recovery analysis demonstrated that the analytical method was adequate to support the hydrolysis study of the test item, providing an indication of the repeatability of the analytical method and of the uniformity of the application procedure for the test item in the Tier 1 test. 

The analytical method was sufficiently sensitive to quantify the test item concentrations down to 10% less than the initial concentration in the Tier tests. 

 

A Preliminary test (Tier 1) was performed: the rate of hydrolysis at 50 ± 0.2°C was assessed for the test item at nominal fortified concentration of 20.6166 mg/L in pH 4, pH 7 and pH 9 buffers solutions. 
The preliminary test results revealed that the rate of hydrolysis (percent degradation) of the test item after 5 days of incubation at 50 ± 0.2°C was 93.47% in pH 4 buffer, 91.95% in pH 7 buffer and 93.50% in pH 9 buffer solutions respectively. Based on the Tier 1 test results, the test item was considered as hydrolytically unstable as more than 10% hydrolysis was obtained and no formation of breakdown products was observed in pH 4, pH 7 and pH 9 buffers at 50 ± 0.2°C after 5 days. 

Hence, further testing of Tier 2 was conducted for the test item in pH 4, pH 7 and pH 9 at nominal fortified concentration of 20.5810 mg/L with solutions being maintained at three different temperatures of 20.0 ± 0.5°C, 35.0 ± 0.5°C and 50.0 ± 0.5°C at various time periods.
The mean logarithms of the relative concentrations were plotted against time using linear regression analysis and  the rate constant and half-life (DT50) were calculated from the Tier 2 results. Using the experimentally determined kobs for each pH and temperature, the temperature dependency was calculated using the relation ln K vs 1/T.

With the regression parameters, by use of the Arrhenius relationships for acid, neutral and base catalysed hydrolysis, pseudo first-order rate constants Kobs (total), and thus half-lives were calculated for hydrolytically behavior of the test item at 25°C. Kobs (total) was calculated as sum of the experimentally determined rate constant values.

At pH 4, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.1123 hour-1 and 6.17 hours, respectively.
At pH 7, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.0572 hour-1 and 12.12 hours, respectively.
At pH 9, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.0715 hour-1 and 9.69 hours, respectively.
At pH 4, 7 and 9, the total rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.2409 hour-1 and 2.88 hours, respectively.

The pH of each of the test solutions was determined at all the intervals. The results show that there were no significant changes in the pH values in all the test solutions of respective buffers. The results show that the hydrolytic degradation of the test item under sterile conditions strongly depends on the temperature and the pH value. Based on Tier 2 test chromatograms, no breakdown products were detected, hence Tier 3 test for the identification of other products was not performed.

Sterility was checked at higher kinetic test tier 2 experimental start 0th Hour (before incubation) in all the test systems and at experiment termination as follows: 20th Hour in pH 4, pH 7 and pH 9 buffer solutions incubated at 50°C, at 24th Hour in pH 4, pH 7 and pH 9 buffers at 35°C, at 24th Hour in pH 4 buffer at 20°C and at 96th Hour in pH 7 and pH 9 buffers at 20 °C respectively.
During the sterility check, no microbial growth (bacteria) was observed in pH 4, pH 7 and pH 9 samples, respectively at the start and termination of the study.

Description of key information

The hydrolysis of AP 729 as a function of pH in Sterile Aqueous Buffer Solutions of pH 4, pH 7 and pH 9 was studied according to OECD Guideline 111 and was determined by analyzing the analyte concentration at the predetermined intervals using a validated HPLC method.

A Preliminary test (Tier 1) was performed: the rate of hydrolysis at 50 ± 0.2°C was assessed for the test item at nominal fortified concentration of 20.6166 mg/L in pH 4, pH 7 and pH 9 buffers solutions. 
The preliminary test results revealed that the rate of hydrolysis (percent degradation) of the test item after 5 days of incubation at 50 ± 0.2°C was 93.47% in pH 4 buffer, 91.95% in pH 7 buffer and 93.50% in pH 9 buffer solutions respectively. Based on the Tier 1 test results, the test item was considered as hydrolytically unstable as more than 10% hydrolysis was obtained and no formation of breakdown products was observed in pH 4, pH 7 and pH 9 buffers at 50 ± 0.2°C after 5 days. 

In Tier 2 test, the hydrolysis of the test item was investigated as a function of pH (4, 7 and 9) at three temperatures of 20 ± 0.5°C, 35 ± 0.5°C and 50 ± 0.5°C. The mean logarithms of the relative concentrations were plotted against time using linear regression analysis and  the rate constant and half-life (DT50) were calculated from the Tier 2 results. Using the experimentally determined kobs for each pH and temperature, the temperature dependency was calculated using the relation ln K vs 1/T. With the regression parameters, by use of the Arrhenius relationships for acid, neutral and base catalysed hydrolysis, pseudo first-order rate constants Kobs (total), and thus half-lives were calculated for hydrolytically behavior of the test item at 25°C. Kobs (total) was calculated as sum of the experimentally determined rate constant values.

At pH 4, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.1123 hour-1 and 6.17 hours, respectively.
At pH 7, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.0572 hour-1 and 12.12 hours, respectively.
At pH 9, the rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.0715 hour-1 and 9.69 hours, respectively.
At pH 4, 7 and 9, the total rate constant and half-life of the reactions at 25ºC were calculated and found to be 0.2409 hour-1 and 2.88 hours, respectively.

Key value for chemical safety assessment

Half-life for hydrolysis:

2.88 h

at the temperature of:

25 °C