5-bromo-1,3-dichloro-2-fluorobenzene

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Sampling intervals: triplicate sampling at days 0, 1 and 5
Sampling procedures: completed, treated samples were removed at each sampling time
Sample storage: all samples were analysed on the day of sampling

Buffers:

pH 4: 0.01 M, Citric acid/sodium citrate prepared in ultra pure water and sterilized by autoclaving
pH 7: 0.01 M, Dihydrogenphosphate/Hydrogenphosphate prepared in ultra pure water and sterilized by autoclaving
pH 9: 0.01 M, Boric acid/sodium borate prepared in ultra pure water and sterilized by autoclaving

Details on test conditions:

Preparation of test systems
Autoclaving of bufferf: twice for about 30 minutes at 121 °C. After autoclaving, the pH of the solutions was checked and adjusted with sterile base or acid.
Cleansing of vial caps, centrifuge vessels and foam stoppers: rinsing with ethanol:water (7:3) before use. Sterilized material was kept in a sterile bench until use. All treatments were performed in a sterile bench under laminar flow conditions.
Size and type of vessels: 15 mL (25 mL for the 0 DAT samples) amber glass vessels equipped with ground glass stoppers.
1 hr (application control) samples: each vessel had a total volume of 9 mL (6 mL acetonitrile and 3 mL of the respective treatment solution).
0 DAT samples: each vessel had a total volume of 18 mL (6 mL of the respective buffer and 3 mL of the respective treatment solution and 9 mL acetonitrile).
1 DAT and 5 DAT samples: each vessel had a total volume of 9 mL (6 mL of the respective buffer and 3 mL of the respective treatment solution).
Preparation of treatment solution
Three treatment solutions, one in each of the used buffers, were prepared. Saturated solutions of the test item were prepared in sterilized stainless steel centrifuge vessels by mixing excess amounts (pH 4: 90.1 mg, pH 7: 75.8 mg and pH 9: 77.0 mg) of test substance with 250 mL of sterilized buffer. The mixtures were shaken for 30 min and centrifuged in an ultra centrifuge at 14 000 rpm. Afterwards 100 mL of the resulting solutions taken from the top layer in the centrifuge vessels were removed and used as treatment solutions. The concentrations of the treatment solutions were 18.6 mg/L (pH 4) and 17.6 mg/L (pH 7 and pH 9).
Application of test substance
All manipulations were performed on a Nunc Safe Flow 1.8 sterile bench.
Triplicate test vessels with 6 mL of buffer solution were prepared for each test temperature (50 °C and ambient temperature) and each sampling time (0 DAT, 1 DAT and 5 DAT).
The 0 DAT samples had the 6 mL buffer solution directly mixed with 9 mL of acetonitrile in order to prevent test item losses by volatilization after application. The test vessels were then treated with 3 mL of the respective treatment solution using a Gilson pipette.
The 1 DAT and 5 DAT samples had the 6 mL buffer solution treated with 3 mL of the respective treatment solution using a Gilson pipette. The samples had a foam stopper inserted into the neck of the vessels (see section 2.1) immediately after application. Any direct contact between the foam stopper and the applied buffer solution was avoided.
Report Number: SYN-077/1-35 Page 17 of 92
The application control samples, in triplicate, were prepared by applying 3 mL of the respective treatment solutions to 6 mL of acetonitrile. The determined concentrations of the application control solutions were used to calculate the test item % values for each pH at each time point and at each temperature.
Incubation, monitoring of temperature and pH measurement
For the tests carried out, samples were maintained at 50 ºC in a thermostated water bath and at ambient temperature in the fume hood (21 °C), in the dark. The temperature was monitored using a data logger recording continuously throughout the incubation period. The pH of the buffer solutions was determined in control samples before and after the incubation.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

6.2 mg/L

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

5.86 mg/L

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

5.88 mg/L

Number of replicates:

Three per buffer

Positive controls:

no

Negative controls:

yes

Statistical methods:

Not applicable

Preliminary study:

For the test at ambient temperature (21 °C), the average recoveries ranged from 98.0% to 118.2% of the applied for all pH values. The test item was fully recovered in all individual samples. In two samples the recovery exceeded 120%. The reason are most probably experimental errors caused by the unusual test substance properties. In addition it should be stated that the majority of the test item (>90% of applied) for all samples at 1 DAT and 5 DAT was found in the extracts of foam stoppers, indicating nearly complete volatilization.
For the test at 50 °C, the average recoveries ranged from 91.7% to 108.1% of the applied for all pH values. The test item was fully recovered in all individual samples. In one sample the recovery was below 80% which was excluded as an outlier due to the foam touching the aqueous phase during sample work-up (indentified as contaminated sample). After 1 day and 5 days of incubation the entire test item was recovered from the foam stoppers.

Test performance:

The test substance was fully recovered from the test samples at all three sampling time points (0, 1, and 5 DAT) and both test temperatures.

Transformation products:

no

% Recovery:

0

pH:

4

Temp.:

50 °C

Duration:

1 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Due to its volatile nature, the substance evaporated from the aqueous test solution very rapidly at 50 °C. A fraction of 0% was found in the aqueous phase after 1 day (and of 0% after five days), whereas a fraction of 92% was recovered after 1 day from the foam stoppers used as a trap for volatilised substance.

% Recovery:

10.1

pH:

4

Temp.:

20 °C

Duration:

1 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Due to its volatile nature, the substance evaporated rapidly from the aqueous test solution leading in low recovery in the aqueous phase. A fraction of 96.1% was recovered after 1 day from the foam stoppers used as a trap for volatilised substance.

% Recovery:

2.6

pH:

7

Temp.:

50 °C

Duration:

1 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Due to its volatile nature, the substance evaporated from the aqueous test solution very rapidly at 50 °C. A fraction of 2.6% was found in the aqueous phase after 1 day (and of 0% after five days), whereas a fraction of 96.6% was recovered after 1 day from the foam stoppers used as a trap for volatilised substance.

% Recovery:

11.1

pH:

7

Temp.:

20 °C

Duration:

1 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Due to its volatile nature, the substance evaporated rapidly from the aqueous test solution leading in low recovery in the aqueous phase. A fraction of 98.8% was recovered after 1 day from the foam stoppers used as a trap for volatilised substance.

% Recovery:

0.9

pH:

9

Temp.:

50 °C

Duration:

1 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Due to its volatile nature, the substance evaporated from the aqueous test solution very rapidly at 50 °C. A fraction of 0.9% was found in the aqueous phase after 1 day (and of 0% after five days), whereas a fraction of 91.7% was recovered after 1 day from the foam stoppers used as a trap for volatilised substance.

% Recovery:

10.1

pH:

9

Temp.:

20 °C

Duration:

1 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

Due to its volatile nature, the substance evaporated rapidly from the aqueous test solution leading in low recovery in the aqueous phase. A fraction of 96.1% was recovered after 1 day from the foam stoppers used as a trap for volatilised substance.

Key result

Remarks on result:

hydrolytically stable based on preliminary test

Details on results:

The substance evaporated rapidly from the aqueous test phase under the conditions of two preliminary tests performed at 50 °C and at room temperature. The substance could be fully recovered in the extracts from foam stoppers that were used as a trap for the volatilised substance in the test vessels.

Validity criteria fulfilled:

yes

Conclusions:

The hydrolysis rate of degradation of the substance could not be assessed at pH 4, 7 and 9 at 50 °C (Tier 1 test) or at ambient temperature due to the high volatility of the test substance under the aqueous (sterile) conditions.
The substance was found to quickly volatilise from the aqueous buffer phase and was captured in the enclosed foam stoppers within the test vessels during the course of the experiment.
No degradation of the substance was observed under the test conditions at 50 °C and at ambient temperature over the 5 days.

Executive summary:

The hydrolysis of the substance at approximately 6 mg/L was investigated under GLP and to OECD TG 111 in the dark in sterile aqueous buffered solutions at pH 4 [6.2 mg/L], pH 7 [5.9 mg/L] and pH 9 [5.9 mg/L] at 50 °C (Tier 1 test) and at ambient temperature (21 °C) as a control for up to 5 days.

Due to the aqueous volatility of the test substance, the test design was modified by the introduction of foam stoppers into the incubation vessels serving as adsorption traps for the volatilized test item.

Triplicate samples from each pH were analysed at 0 DAT, 1 DAT and 5 DAT incubation at 50 °C and at ambient temperature. The aqueous solutions were analysed after dilution with acetonitrile (1:1). The foam stoppers were extracted with acetonitrile and the acetonitrile extracts were diluted 1:1 with water and analysed directly by high performance liquid chromatography with UV-detection.

The application rate was determined by separate application control samples for each pH value. The application control samples were prepared by applying the dosing solution in the respective pH buffer into acetonitrile followed by immediate analysis (approx. 1 hr).

Only mean recovery values of 10.1-11.1% and <LOQ of the substance remained in the aqueous phase after 1 DAT at ambient and 50 ºC temperatures respectively. After 5 DAT the aqueous phase contained mean recovery values of 0% of the substance for both temperatures and all pHs. All of the volatilized substance was accounted for in the foam stopper extraction solutions.

The total mean recovery of the substance was 108.7% (range 107.6% to 110.8%) for the ambient temperature test and 98.3% (range 94.5% to 102.5%) for the 50 °C test. Hence, HPLC analysis confirmed that the substance had not degraded during this study and no obvious losses from the test system had taken place.

Therefore, due to the volatile nature of the substance under the aqueous (sterile) conditions at both ambient temperature and at 50 °C, the hydrolysis of the substance could not be assessed.

The temperatures remained constant throughout the incubation periods (50 ± 0.5 °C and 21 ± 1 °C) and there was no significant variation in the pH values of the buffered solutions. The samples also remained sterile throughout the study.

Description of key information

No hydrolysis occurred at 50 °C at pH 4, 7 or 9 (GLP, OECD TG 111)

Additional information

Due to the volatility of the substance from the aqueous test solutions at ambient temperature and at a test temperature of 50 °C, a stable aqueous solution of the test substance could not be maintained over the period of the screening study of five days. The majority of the test substance was collected in polyurethane foam stoppers that were placed in the test vessels to trap evaporating test material. The mass balance obtained by analysing the test substance in the aqueous, buffered test solutions and in extracts of the polyurethane foam stoppers resulted in full recovery of the test substance, demonstrating that no degradation of the test substance occurred in the test system over the test period of five days under the applied test conditions.