3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2017 to 15 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Preliminary Test- Tier 1
- The concentration of the test material in the test samples was determined immediately after preparation (t=0) and after 5 days. The samples taken at t=5 days were cooled to room temperature using running tap water. The samples were diluted in a 1:1 (v:v) ratio with methanol and analysed.
- Blank buffer solutions containing a similar content of blank spiking solution were treated similarly as the test samples and analysed at t=0.
- The pH of each of the test solutions (except for the blanks) was determined at each sampling time.

Main study - Tier 2
- The concentrations of the test item were determined immediately after preparation (t=0) and at several sampling points after t=0.
- Blank buffer solutions were treated similarly as the test samples and analysed at t=0.
- The pH of each of the test solutions (except for the blanks) was determined at least at the beginning and at the end of the test.

Buffers:

- Acetate buffer pH 4, 0.01 M: A solution of 16.7 % 0.01 M sodium acetate in water and 83.3 % 0.01 M acetic acid in water. The buffer contains 0.0009 % (w/v) sodium azide.
- Phosphate buffer pH 7, 0.01 M: A solution of 0.01 M potassium di-hydrogenphosphate in water adjusted to pH 7 using 1 N sodium hydroxide. The buffer contains 0.0009 % (w/v) sodium azide.
- Borate buffer pH 9, 0.01 M: A solution of 0.01 M boric acid in water and 0.01 M potassium chloride in water adjusted to pH 9 using 1 N sodium hydroxide. The buffer contains 0.0009 % (w/v) sodium azide.

Details on test conditions:

Preliminary test - Tier 1
- The buffer solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. The test material was spiked to the solutions at a target concentration of 2 mg/L using a spiking solution in methanol. - For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 50.1 ± 0.1°C.

Main study - Tier 2
- Test samples were prepared and treated similarly as during the preliminary test.

Duration:

5 d

pH:

4

Temp.:

25 °C

Duration:

5 d

pH:

7

Temp.:

20 °C

Duration:

5 d

pH:

7

Temp.:

25 °C

Duration:

5 d

pH:

7

Temp.:

50 °C

Duration:

5 d

pH:

7

Temp.:

70 °C

Duration:

5 d

pH:

9

Temp.:

20 °C

Duration:

5 d

pH:

9

Temp.:

25 °C

Duration:

5 d

pH:

9

Temp.:

50 °C

Duration:

5 d

pH:

9

Temp.:

60 °C

Positive controls:

no

Negative controls:

yes

Preliminary study:

- A degree of hydrolysis of ≥ 10 % was observed at pH 7 and pH 9 after 5 days. According to the guideline, the higher Tier test was required to determine the half-life time of the test material.
-A degree of hydrolysis of < 10 % was observed at pH 4 after 5 days. It demonstrated that the half-life time of the test material at 25 °C is > 1 year. According to the guideline, no further tests were required.
- No test material was detected in the blank buffer solutions.
- The mean recoveries of the of the test material containing buffer solutions at t=0 fell within the criterion range of 90 to 110 %. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test material.

Test performance:

- The mean recoveries of the of the test material containing buffer solutions at t=0 fell within the criterion range of 90 to 110 %. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test material.

Transformation products:

no

Details on hydrolysis and appearance of transformation product(s):

Research to investigate the identity or nature and rates of formation and decline of hydrolysis products was not required since the studies were conducted according to the testing guidelines provided in Annex VIII (volume 10-100 tons/year).

% Recovery:

103

pH:

4

Temp.:

25 °C

Duration:

5 d

% Recovery:

99

pH:

7

Temp.:

25 °C

Duration:

5 d

% Recovery:

94

pH:

9

Temp.:

25 °C

Duration:

5 d

% Recovery:

102

pH:

7

Temp.:

20 °C

Duration:

5 d

% Recovery:

97

pH:

7

Temp.:

50 °C

Duration:

5 d

% Recovery:

103

pH:

7

Temp.:

70 °C

Duration:

5 d

% Recovery:

95

pH:

9

Temp.:

20 °C

Duration:

5 d

% Recovery:

97

pH:

9

Temp.:

50 °C

Duration:

5 d

% Recovery:

96

pH:

9

Temp.:

60 °C

Duration:

5 d

Key result

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0 h-1

DT50:

266 d

Type:

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

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

187 d

Type:

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

Key result

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.001 h-1

DT50:

29 d

Type:

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

Key result

pH:

7

Temp.:

70 °C

Hydrolysis rate constant:

0.009 h-1

DT50:

76 h

Type:

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

Key result

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.002 h-1

DT50:

324 h

Type:

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

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.004 h-1

DT50:

168 h

Type:

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

Key result

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.081 h-1

DT50:

8.6 h

Type:

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

Key result

pH:

9

Temp.:

60 °C

Hydrolysis rate constant:

0.219 h-1

DT50:

3.2 h

Type:

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

Details on results:

Main study - Tier 2
pH 7
- The pH was in the range 7.0 – 7.1. No test material was detected in the blank buffer solutions. The mean recoveries of the test material containing buffer solutions at t=0 fell within the criterion range of 90 to 110 %. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test material.
- For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10 and 90 % were plotted against time. At all temperatures linear relationships were obtained, except for 20 °C.
- The half-life times of the test material were determined according to the model for pseudo-first order reactions. All logarithms of the relative concentrations were correlated with time using linear regression analysis.
- The rate constant (kobs) and half-life time of the test material at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25 °C.

pH 9
- The pH was in the range 9.0 – 9.1. No test material was detected in the blank buffer solutions.
- The mean recoveries of the test material containing buffer solutions at t=0 fell within the criterion range of 90 to 110 %. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test material.
- For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10 and 90% were plotted against time. At all temperatures linear relationships were obtained.
- The half-life times of the test material were determined according to the model for pseudo-first order reactions. All logarithms of the relative concentrations were correlated with time using linear regression analysis.
- The rate constant (kobs) and half-life time of the test item at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25 °C.

Table 1: Summary of half-times of the test material

pH 4		pH 7		pH 9
Temperature (°C)	t1/2	Temperature (°C)	t1/2	Temperature (°C)	t1/2
25	>1 year	20	266 d	20	324 h
		25	187 d	25	168 h
		50	29 d	50	8.6 h
		70	76 h	60	3.2 h

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the half life of the test material at pH 4 was > 1 year at 25 °C. At pH 7 the half life values were; at 20 °C: 266 days, at 25 °C: 187 days, at 50 °C: 29 days and at 70 °C: 76 hours. At pH 9 the half life values were; at 20 °C: 324 hours, at 25 °C: 168 hours, at 50 °C: 8.6 hours and at 60 °C: 3.2 hours.

Executive summary:

The hydrolysis as a function of pH of the test material was investigated in accordance with the standardised guidelines OECD 111, EU Method C.7 and EPA OPPTS 835.2120, under GLP conditions.

The rate of hydrolysis of the test item as a function of pH was determined at pH values normally found in the environment (pH 4-9). A first preliminary test supported by UPLC-MS/MS was not successful due to the analytical method. After development and validation of a UPLC-UV method, the preliminary test was repeated.

The buffer solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. The test item was spiked to the solutions at a target concentration of 2 mg/L using a spiking solution in methanol. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 50.1 ± 0.1°C. Test samples were analysed for test material concentration at t=0 and at several following time points during the 5 day period.

In the preliminary test (tier 1) a degree of hydrolysis of ≥ 10 % was observed at pH 7 and pH 9 after 5 days. According to the guideline, the higher Tier test was required to determine the half-life time of the test item. A degree of hydrolysis of < 10% was observed at pH 4 after 5 days. It demonstrated that the half-life time of the test item at 25 °C is > 1 year. According to the guideline, no further tests were required at pH 4.

The analytical results showed that the pH values were within the expected range and also showed that no test item was detected in the blank buffer solutions. Since the mean recoveries of the of the test item containing buffer solutions at t=0 fell within the criterion range of 90 to 110 %. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test item.

Under the conditions of this study the half life of the test material at pH 4 was > 1 year at 25 °C. At pH 7 the half life values were; at 20 °C: 266 days, at 25 °C: 187 days, at 50 °C: 29 days and at 70 °C: 76 hours. At pH 9 the half life values were; at 20 °C: 324 hours, at 25 °C: 168 hours, at 50 °C: 8.6 hours and at 60 °C: 3.2 hours.

Description of key information

Under the conditions of this study the half life of the test material at pH 4 was > 1 year at 25 °C. At pH 7 the half life values were; at 20 °C: 266 days, at 25 °C: 187 days, at 50 °C: 29 days and at 70 °C: 76 hours. At pH 9 the half life values were; at 20 °C: 324 hours, at 25 °C: 168 hours, at 50 °C: 8.6 hours and at 60 °C: 3.2 hours.

Additional information

The hydrolysis as a function of pH of the test material was investigated in accordance with the standardised guidelines OECD 111, EU Method C.7 and EPA OPPTS 835.2120, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The rate of hydrolysis of the test item as a function of pH was determined at pH values normally found in the environment (pH 4-9). A first preliminary test supported by UPLC-MS/MS was not successful due to the analytical method. After development and validation of a UPLC-UV method, the preliminary test was repeated.

The buffer solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. The test item was spiked to the solutions at a target concentration of 2 mg/L using a spiking solution in methanol. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 50.1 ± 0.1°C. Test samples were analysed for test material concentration at t=0 and at several following time points during the 5 day period.

In the preliminary test (tier 1) a degree of hydrolysis of ≥ 10 % was observed at pH 7 and pH 9 after 5 days. According to the guideline, the higher Tier test was required to determine the half-life time of the test item. A degree of hydrolysis of < 10% was observed at pH 4 after 5 days. It demonstrated that the half-life time of the test item at 25 °C is > 1 year. According to the guideline, no further tests were required at pH 4.

The analytical results showed that the pH values were within the expected range and also showed that no test item was detected in the blank buffer solutions. Since the mean recoveries of the of the test item containing buffer solutions at t=0 fell within the criterion range of 90 to 110 %. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test item.

Under the conditions of this study the half life of the test material at pH 4 was > 1 year at 25 °C. At pH 7 the half life values were; at 20 °C: 266 days, at 25 °C: 187 days, at 50 °C: 29 days and at 70 °C: 76 hours. At pH 9 the half life values were; at 20 °C: 324 hours, at 25 °C: 168 hours, at 50 °C: 8.6 hours and at 60 °C: 3.2 hours.