3,4-dihydro-2H-1,4-benzoxazin-6-ol

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-11-16 to 2021-05-18

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

April 13, 2004.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The following total number of samples was prepared:
• Preliminary Test (Tier 1): 8 per pH
• Main Test (Tier 2): 18 per pH and temperature

Sampling Procedure and Schedule:
Preliminary Test (Tier 1)
Test Duration:
The incubation was terminated after 5 days.
Sampling of the Test Samples:
For all three pH levels: 0, 3, 24, 120 h
At each sampling point, samples were taken in duplicate.

Main Test (Tier 2)
Test Duration:
The incubation was terminated after
• pH 4: 30 d (20°C), 30 d (30°C), 21 d (50°C)
• pH 7: 30 d (20°C), 30 d (30°C), 21 d (50°C)
• pH 9: 15 d (20°C), 12 d (30°C), 10 d (50°C)
Sampling of the Test Samples:
• pH 4:
o 20°C: 0, 2, 5, 8, 12, 15, 21, 26, 30 d
o 30°C: 0, 2, 5, 8, 12, 15, 21, 26, 30 d
o 50°C: 0, 6 h // 1, 2, 3, 7, 10, 16, 21 d
• pH 7:
o 20°C: 0, 2, 5, 8, 12, 15, 21, 26, 30 d
o 30°C: 0, 2, 5, 8, 12, 15, 21, 26, 30 d
o 50°C: 0, 6 h // 1, 2, 3, 7, 10, 16, 21 d
• pH 9:
o 20°C: 0, 1, 2, 5, 8, 12, 15 d
o 30°C: 0, 4 h // 1, 2, 5, 8, 12 d
o 50°C: 0, 2, 6 h // 1, 2, 3, 4, 7, 10 d
At each sampling point, samples were taken in duplicate.

After each step of sample preparation a procedural recovery was calculated.
Samples were diluted by a factor of 2 with acetonitrile to prevent further degradation of the test item after sampling.

Buffers:

pH 4: 0.05 M acetate buffer
410 mL acetic acid (0.1 M) was added to 90 mL sodium acetate (0.1 M). The solution was filled up to 1000 mL with pure water.

pH 7: 0.05 M phosphate buffer
296 mL NaOH (0.1 M) was added to 500 mL potassium dihydrogenphosphate (0.1 M). The solution was filled up to 1000 mL with pure water.

pH 9: 0.05 M boric acid buffer
213 mL NaOH (0.1 M) was added to 500 mL boric acid (0.1 M in potassium chloride, 0.1 M). The solution was filled up to 1000 mL with pure water.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: Glass flasks (hermetically closed) were used for the test.
- Sterilisation method: Degased buffer solutions were sterilized by microfiltration (0.2 μm) and the used glassware was sterilised using an autoclave (20 min at 121°C) prior to application. A sterility confirmation test was carried out in course of the study. A commercially available dip slide kit was used for a total count of bacteria and to determine the total count of yeast and moulds.
- Lighting: Darkness
- Measures to exclude oxygen: To avoid oxidation the buffer solutions were purged with inert gas (nitrogen) prior to sterilisation.

TEST MEDIUM
- Preparation of test medium: Sterile aqueous solutions buffered at pH 4, 7 and 9.
The pH of each buffer solution was measured with a calibrated pH meter.
- Renewal of test solution: no
- Identity and concentration of co-solvent: none used
- Stock Solution of the Test Item:
Two individual stock solutions (A and B) were prepared in ACN/pure water (20:80, v/v) with a concentration of 2.00 and 2.01 g/L in case of the preliminary test (Tier 1), 2.00 and 2.01 g/L in case of main test (Tier 2) at 20 and 30°C, and 2.00 g/L in case of main test (Tier 2) at 50°C.
- Application Solution of the Test Item: The stock solutions were used.
The final concentration of the test item in the aqueous phase was below 0.01 M or half of its water solubility and the content of organic was ≤ 1% v/v. Each replicate (A and B) was prepared individually.
Treatment Rate:
Preliminary test (Tier 1): 100 mg/L,
Main test (Tier 2): 100 mg/L

Duration:

5 d

Temp.:

50 °C

Remarks:

Preliminary Test (Tier 1)

Preliminary study:

Immediately after application (0 h) recoveries of the samples were as follows:
• Preliminary test (Tier 1): 100.5-108.3% of the nominal applied concentration. Recoveries of the applied concentration were considered acceptable for the preliminary test (Tier 1). Result evaluation was performed based on the nominal concentration.

Test performance:

Immediately after application (0 h) recoveries of the samples were as follows:
Main test (Tier 2): 109.0-111.9%, 118.2-122.0% and 112.6-115.1% of the nominal applied concentration, for pH 4, 7 and 9 respectively.
In the main test (Tier 2) recoveries were out of the desired range but still below 0.01 M or half of its water solubility. Result evaluation was performed based on the applied concentration (mean concentration at 0 h).

Transformation products:

not measured

pH:

9

Temp.:

50 °C

Duration:

10 d

Remarks on result:

other: At the end of incubation recoveries (mean) were < LOQ (10% of the applied concentration)

pH:

9

Temp.:

30 °C

Duration:

12 d

Remarks on result:

other: At the end of incubation recoveries (mean) were < LOQ (10% of the applied concentration)

pH:

9

Temp.:

20 °C

Duration:

15 d

Remarks on result:

other: At the end of incubation recoveries (mean) were < LOQ (10% of the applied concentration)

pH:

7

Temp.:

50 °C

Duration:

21 d

Remarks on result:

other: LOQ (10% of the nominal applied concentration)

% Recovery:

17.3

pH:

7

Temp.:

30 °C

Duration:

30 d

% Recovery:

36.9

pH:

7

Temp.:

20 °C

Duration:

30 d

% Recovery:

17.1

pH:

4

Temp.:

50 °C

Duration:

21 d

% Recovery:

46.1

pH:

4

Temp.:

30 °C

Duration:

30 d

% Recovery:

65.1

pH:

4

Temp.:

20 °C

Duration:

30 d

pH:

4

Temp.:

20 °C

Hydrolysis rate constant:

0.015 d-1

DT50:

45.9 d

pH:

4

Temp.:

30 °C

Hydrolysis rate constant:

0.029 d-1

DT50:

24.1 d

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.092 d-1

DT50:

7.5 d

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.021 d-1

DT50:

33.1 d

Remarks on result:

other: The hydrolysis rate constant and the corresponding DT50 value was calculated for a temperature of 25°C based on the arrhenius parameters

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0.027 d-1

DT50:

26 d

pH:

7

Temp.:

30 °C

Hydrolysis rate constant:

0.063 d-1

DT50:

11.1 d

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.254 d-1

DT50:

2.7 d

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.041 d-1

DT50:

17 d

Remarks on result:

other: The hydrolysis rate constant and the corresponding DT50 value was calculated for a temperature of 25°C based on the arrhenius parameters

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.262 d-1

DT50:

2.7 d

pH:

9

Temp.:

30 °C

Hydrolysis rate constant:

0.436 d-1

DT50:

1.6 d

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.663 d-1

DT50:

1.1 d

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.332 d-1

DT50:

2.1 d

Remarks on result:

other: The hydrolysis rate constant and the corresponding DT50 value was calculated for a temperature of 25°C based on the arrhenius parameters

Other kinetic parameters:

DT90 (d):
pH4-20°C: 153
pH4-30°C: 79.9
pH4-50°C: 25
pH7-20°C: 86.4
pH7-30°C: 36.8
pH7-50°C: 9.1
pH9-20°C: 8.8
pH9-30°C: 5.3
pH9-50°C: 3.5

Details on results:

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

Executive summary:

The present study investigated the hydrolytic behaviour of Hydroxybenzomorpholine in aqueous solutions buffered at pH 4, 7 and 9 and at three elevated temperature, according to OECD 111 guideline and following GLP. Samples were incubated in the dark.

The preliminary test was first carried out for 5 days at 50°C (Tier 1). At the end of incubation, 19.0 to 55.8% of the applied concentration was found over all pH levels and thus the test item can be stated as hydrolytically unstable.
The main test (Tier 2) was performed at pH 4, 7 and 9, and at 20, 30 and 50°C. For pH 4, the final recoveries (mean) were 65.1%, 46.1% and 17.1% of the applied concentration at 20, 30 and 50°C, respectively. For pH 7, the final recoveries (mean) were 36.9% and 17.3% of the applied concentration at 20 and 30°C, respectively, and < LOQ (10% of the applied concentration) at 50°C. For pH 9, the final recoveries were < LOQ (10% of the applied concentration) at all three temperatures.

Description of key information

The present study investigated the hydrolytic behaviour of Hydroxybenzomorpholine in aqueous solutions buffered at pH 4, 7 and 9 and at three elevated temperature, according to OECD 111 guideline and following GLP. Samples were incubated in the dark.

The preliminary test was first carried out for 5 days at 50°C (Tier 1). At the end of incubation, 19.0 to 55.8% of the applied concentration was found over all pH levels and thus the test item can be stated as hydrolytically unstable.
The main test (Tier 2) was performed at pH 4, 7 and 9, and at 20, 30 and 50°C. For pH 4, the final recoveries (mean) were 65.1%, 46.1% and 17.1% of the applied concentration at 20, 30 and 50°C, respectively. For pH 7, the final recoveries (mean) were 36.9% and 17.3% of the applied concentration at 20 and 30°C, respectively, and < LOQ (10% of the applied concentration) at 50°C. For pH 9, the final recoveries were < LOQ (10% of the applied concentration) at all three temperatures.