2,2-Difluoroethyl Acetate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 February 2016 to 19 May 2016

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:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
For the Tier 1 (preliminary test) : at the start and termination of the test i.e. 5 days for each pH (4 , 7 and 9).
For the Tier 2 (definitive test):
at pH 9.0 and 20°C: 0, 0.5, 1, 2, 3 and 7 day
at pH 9.0 and 35°C: 0, 0.125, 0.25, 0.5, 1 and 2 day
at pH 9.0 and 50°C: 0, 0.0625, 0.125, 0.25, 0.5 and 1 day
at pH 7.0 and 20°C: 0, 3, 7, 14, 21 and 32 day
at pH 7.0 and 35°C: 0, 3, 7, 14, 21 and 32 day
at pH 7 .0 and 50°C: 0, 1, 2, 3, 7 and 14 day
A detailed sampling schedule is presented in Table 1 in "Any other information on materials and methods incl. tables".

- Sampling method and equilibration before analysis:
Headspace Sampling: For HS/GC/MS analysis, an aliquot (2.0 mL) of each test sample was withdrawn using a 2.50 mL graduated glass syringe into 20 mL amber vial and capped immediately with a screw cap containing a PTFE-lined septum for equilibration.
Equilibration: The vials were equilibrated in a VWR temperature-controlled incubator at approximately 37 ºC on a VWR mini shaker set at 250 rpm for at least 15 minutes before 250 μL aliquots of the headspace were sampled using a 500-μL graduated glass gas syringe for HS GC/MS analysis. The syringe was flushed with air between injections.

- Sampling intervals/times for pH measurements:
For the Tier 1 (preliminary test): at the start and termination of the test i.e. 5 days for each pH (4 , 7 and 9).
For the Tier 2 (definitive test): at the start and termination of the test for each pH and temperature conditions.

- Sampling intervals/times for sterility check:
For the Tier 2 (definitive test): at the termination of the test for each pH and temperature conditions.

Buffers:

- pH: 4, 7 and 9
- Type and final molarity of buffer: 0.01M for pH 4.0, pH 7.0 and pH 9.0
- Composition of buffer: Sterile aqueous pH buffered solutions at pH 4 (acetate), pH 7 (phosphate) and pH 9 (borate) (± 0.1 pH unit) were prepared by dissolving the appropriate acid and bases in HPLC grade water using an analytical balance and graduated cylinders. The total salt concentration in each pH buffered solution preparation did not exceed 0.01 M. Immediately following preparation, the pH of each solution was measured and was then adjusted to the final (target) pH with small additions of acid or base. The pH of each prepared buffered solution was verified to be within ±0.1 pH unit of the target nominal using a pH meter (Orion Star A111) at room temperature with an accuracy of ±0.01 pH unit.
The preparation of buffered solutions at each of the target pH conditions was as follows:
> 0.01M pH 4.0: Buffer solution was prepared by dissolving 0.1231 g of sodium acetate and 480 μL of acetic acid in 1000 mL of HPLC water. The pH of the resulting solution was 4.02.
>0.01M pH 7.0: Buffer solution was prepared by dissolving 0.6804 g of potassium dihydrogen phosphate and 0.8535 g of dipotassium hydrogen-phosphate in 1000 mL of HPLC water. The pH of the resultant buffer solution was 7.05 which was further adjusted to 7.0 using a 1M aqueous H3PO4 solution.
>0.01M pH 9.0: Buffer solution was prepared by dissolving 0.6183 g of boric acid in 1000 mL of HPLC water. The pH of the resultant buffer solution was adjusted to 9.02 using 1.5N KOH solution.
Each prepared pH buffered solution was stored at refrigerator temperature in a separate 1-L amber glass bottle. Prior to use of a given pH buffered solution, a portion of the bulk preparation was removed.

Details on test conditions:

TEST SYSTEM:

- Type, material and volume of test flasks, other equipment used:
The test vessels were autoclaved approximately 14-mL amber vials (Tier 1) and 14.8-mL amber serum vials (Tier 2) with aluminum open top crimp caps fitted with PTFE-lined silicone septa. The vials were Wheaton 10-mL amber serum vials (Cat# 06-402B, actual capacity: approximately 14 mL) and Electron Microscopy Science 10-mL amber serum vials (Cat #: 50-366-780, actual capacity: approximately 14.8 mL).
The test vessels for headspace GC/MS analysis were 20-mL amber glass vials with open-top white polypropylene caps and PTFE/silicone septa from Fisher Scientific (Thermo Scientific™ 33920A, actual capacity: approximately 23.5 mL).

- Sterilisation method: The buffers for the Tier 1 study were autoclaved at approximately 121 ºC at 15 psi for 30 minutes. Prior to the use of the pH 7.0 and pH 9.0 buffers in the Tier 2 study, the buffers were further degassed and sterilized by vacuum filtration through 0.2-μm filters (Corning/430515).

- Measures to exclude oxygen: The buffers were purged with sterile nitrogen for at least 5 minutes to exclude oxygen before dosing.

TEST MEDIUM

The hydrolysis study was conducted using sterile aqueous pH buffered solutions at pH 4 (acetate), pH 7 (phosphate) and pH 9 (borate) (± 0.1 pH unit) prepared as detailed in the section above entitled "Buffers".

OTHER DETAILS ON TEST PROCEDURE:

- Preparation of test solutions:
For Tier 1, the preparation of the test solution was not described.
For Tier 2, for each of the pH 7 and pH 9 test vessels, pH buffered solution was fortified at a target nominal 2,2-Difluoroethyl acetate concentration of 4 µg/mL on Day 0 of the test. Test samples were prepared by completely filling approximately 14.8 mL pre-labeled amber vials with respective buffer solution using a 25 mL sterile graduated glass pipet. Each vial was capped with an aluminum crimp cap containing a PTFE-lined silicone septum. The exact weight of the buffer in each vial was calculated as the difference of the weight of the test vial (plus cap) before and after the addition of the buffer. An aliquot (74 μL) of the buffer was removed from each vial and then 74 μL of a stock solution (800 μg/mL 2,2-Difluoroethyl acetate) was added using a 100 μL glass syringe. A total of 42 test samples were prepared for each pH buffer. Vials were placed on a Multitube Vortexer (Fisher Scientific) with the speed set at 8 for 10 min and then incubated at 20, 35 and 50 °C for various intervals of time before sampling for HS GC/MS analysis.

- Processing of replicates at different sampling times:
Following initial sampling at day 0, two samples at each pH and temperature test condition were removed from the temperature-controlled incubator for processing at the selected intervals. At each sampling interval, test vials were allowed to cool to ambient temperature in a water bath and then sampled for headspace analysis. The pH and sterility were measured and recorded at the test termination of each pH test condition using one of the incubated hydrolysis samples, respectively. At least one incubation sample at each pH and temperature condition remained at the conclusion of each incubation period. These samples were extra preparations (i.e. in case of breakage/spillage) which were not required to complete the study and were therefore not processed.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 1.

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 1.

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 1.

Duration:

14 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 2.

Duration:

32 d

pH:

7

Temp.:

35 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 2.

Duration:

32 d

pH:

7

Temp.:

20 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 2.

Duration:

1 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 2.

Duration:

2 d

pH:

9

Temp.:

35 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 2.

Duration:

7 d

pH:

9

Temp.:

20 °C

Initial conc. measured:

ca. 4 mg/L

Remarks:

Nominal concentration. Tier 2.

Number of replicates:

For the Tier 1 (preliminary test): For each pH (i.e. 4.0, 7.0 and 9.0), triplicate samples at each measurement time.
For the Tier 2 (definitive test): For each pH (i.e. 4.0, 7.0 and 9.0) and temperature (20, 35 and 50°C) condition, duplicate samples at each mesurement time.
One extra sample from each pH and 20 °C was used for pH analysis at the experimental start and one extra sample from each pH and temperature was used for pH and sterile analysis at the experimental termination.

Preliminary study:

The Preliminary test (Tier 1) was performed by incubation of the test substance at 50 °C for five days in pH 4, 7 and 9 buffers. The preliminary test results showed that the test substance degraded rapidly at pH 9 (97.9% degradation), more slowly at pH 7 (83.4% degradation) and was stable (15.4% degradation) at pH 4 (Tables 4, 5 and 6). From the hydrolysis mechanism for 2,2-difluoroethyl acetate, 2,2-Difluoroethanol was considered to be the likely principal degradant. The degradation percentage of the 2,2-difluoroethyl acetate in the test vessels at pH 4, after normalization, was 3.9% (84.6/(84.6 + 3.4)*100), i.e., less than 10% degradation (See Table 6 below).

Test performance:

ANALYTICAL METHOD PERFORMANCE (QC SAMPLE):
- For the Tier 1:
The mean QC recoveries study ranged during the five days of the study (Table 3):
For the pH4, from 94.2 to 95.3% for the test substance (2,2-Difluoroethyl acetate) and 89.8 to 90.7% for 2,2-Difluoroethanol.
For the pH 7, from 92.2 to 93.4% for the test substance (2,2-Difluoroethyl acetate) and 88.0 to 91.6% for 2,2-Difluoroethanol.
For the pH 9, from 69.8 to 82.3% for the test substance (2,2-Difluoroethyl acetate) and 100.8 to 107.7% for 2,2-Difluoroethanol.

- For the Tier 2:
The mean QC recoveries for the pH 9 study ranged from 82.3 to 88.5% for the test substance (2,2-Difluoroethyl acetate) and 89.1 to 97.1% for 2,2-Difluoroethanol during the seven days of the study (Table 7).
The mean QC recoveries for the pH 7 study ranged from 82.1 to 102.4% for the test substance (2,2-Difluoroethyl acetate) and 85.3 to 102.4% for 2,2-Difluoroethanol during the thirty-two days of the study (Table 8).

Transformation products:

yes

No.:

#1

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

1) Hydrolysis Potential of 2,2-Difluoroethyl acetate:
At pH 9.0:
- The average amount of 2,2-Difluoroethyl acetate in the pH 9.0 hydrolysis samples incubated at 20 ºC decreased from 96.6% at Day 0 to 4.2% at Day 7 (Table 9).
- The average amount of 2,2-Difluoroethyl acetate in the pH 9.0 hydrolysis samples incubated at 35 ºC decreased from 96.6% at Day 0 to 1.4% at Day 2 (Table 10).
- The average amount of 2,2-Difluoroethyl acetate in the pH 9.0 hydrolysis samples incubated at 50 ºC decreased from 96.6% at Day 0 to 0.9% at Day 1 (Table 11).

At pH 7.0:
- The average amount of 2,2-Difluoroethyl acetate in the pH 7.0 hydrolysis samples incubated at 20 ºC decreased from 114.5% at Day 0 to 62.8% at Day 32 (Table 12).
- The average amount of 2,2-Difluoroethyl acetate in the pH 7.0 hydrolysis samples incubated at 35 ºC decreased from 114.5% at Day 0 to 11.7% at Day 32 (Table 13).
- The average amount of 2,2-Difluoroethyl acetate in the pH 7.0 hydrolysis samples incubated at 50 ºC decreased from 114.5% at Day 0 to 1.0% at Day 14 (Table 14).

2) Transformation product 2,2-difluoroethanol (DFE):
At pH9:
The average amount of DFE in the pH 9.0 hydrolysis samples incubated at 20 ºC increased from 10.7% at Day 0 to 92.4% at Day 7 (Table 9).
The average amount of DFE in the pH 9.0 hydrolysis samples incubated at 35 ºC increased from 10.7% at Day 0 to 96.6% at Day 2 (Table 10).
The average amount of DFE in the pH 9.0 hydrolysis samples incubated at 50 ºC increased from 10.7% at Day 0 to 89.9% at Day 1 (Table 11).

At pH 7.0 :
The average amount of DFE in the pH 7.0 hydrolysis samples incubated at 20 ºC increased from The average amount of DFE in the pH 7.0 hydrolysis samples incubated at 35 ºC increased from The average amount of DFE in the pH 7.0 hydrolysis samples incubated at 50 ºC increased from

% Recovery:

84.6

pH:

4

Temp.:

50 °C

Duration:

5 d

% Recovery:

1

pH:

7

Temp.:

50 °C

Duration:

14 d

% Recovery:

11.7

pH:

7

Temp.:

35 °C

Duration:

32 d

% Recovery:

62.8

pH:

7

Temp.:

20 °C

Duration:

32 d

% Recovery:

0.9

pH:

9

Temp.:

50 °C

Duration:

1 d

% Recovery:

1.4

pH:

9

Temp.:

35 °C

Duration:

2 d

% Recovery:

4.2

pH:

9

Temp.:

20 °C

Duration:

7 d

Key result

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Key result

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.34 d-1

DT50:

2 d

Type:

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

Key result

pH:

7

Temp.:

35 °C

Hydrolysis rate constant:

0.067 d-1

DT50:

10.3 d

Type:

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

Key result

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0.015 d-1

DT50:

46.8 d

Type:

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

Key result

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

12.917 d-1

DT50:

0.05 d

Type:

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

Key result

pH:

9

Temp.:

35 °C

Hydrolysis rate constant:

2.464 d-1

DT50:

0.28 d

Type:

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

Key result

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.436 d-1

DT50:

1.59 d

Type:

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

Details on results:

TEST CONDITIONS:
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
The measured pH of the various buffers at the start and termination of the Tier 1 and Tier 2 studies are shown in Table 2.
Plate count sterility tests were performed on a test sample vial at each test termination during Tier 2. Negative results were observed in all selected test samples.

MASS BALANCE:
The average material balance of test substance for the pH 9 hydrolysis in 20, 35 and 50 °C test system throughout the study ranged from 85.5 to 107.3%, 93.6 to 107.5% and 86.7 to 107.3% of the applied dose, respectively. A summary of the mean material balance as percent of applied dose for the samples of each test system (20, 35 and 50 ºC) is presented in Tables 9 through Table 11.
The average material balance of test substance for the pH 7 hydrolysis in 20, 35 and 50 °C test system throughout the study ranged from 82.4 to 114.8%, 85.4 to 114.8% and 80.3 to 114.8% of the applied dose, respectively. A summary of the mean material balance as percent of applied dose for the samples of each test system (20, 35 and 50 ºC) is presented in Tables 12 through Table 14.

RATE CONSTANT AND HALF-LIFE:
A first-order linear regression analysis was performed on the 20 °C, 35 °C and 50 °C data for the sterile pH 9.0 and pH 7.0 (Tables 15 and 16) to determine the rates of hydrolysis. The DT50 (half-life) values for 2,2-Difluoroethyl acetate under the hydrolysis conditions employed using first-order kinetics are summarized in the " Dissipation DT50 of parent compounds"

ARRHENIUS EQUATION ESTIMATION OF THE DT50 AT 25°C:
The half-life of the test substance at 25 ºC was calculated to be 0.9 days at pH 9.0 and 28 days at pH 7.0 (Table 17).

Table 2: Summary of pH measurements

Tier 2
Sample	Sampling Interval (Days)	Temperature ( °C)	Measured pH
pH
7	0	ambient (20.5 – 23.5)	7.1
	32	20	7.0
	32	35	7.1
	14	50	7.0
9	0	ambient (20.5 – 23.5)	9.0
	7	20	9.0
	2	35	9.0
	1	50	9.0
Tier 1
Sample	Sampling Interval (Days)	Temperature ( °C)	Measured pH
pH
4	0	ambient (20.5 – 23.8)	4.0
	5	50	4.0
7	0	ambient (20.5 – 23.8)	7.0
	5	50	7.0
9	0	ambient (20.5 – 23.8)	9.0
	5	50	8.9

 

Table 3: Concentrations and recovery of 2,2-Difluoroethyl acetate and 2,2-Difluoroethanol (DFE) quality control samples in aqueous pH buffered solutions analyzed by HS/GC/MS during tier 1 test

Sampling Interval (days)	Sample ID	2,2-Difluoroethyl acetate				DFE
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (CV)	Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (CV)
	pH 4 buffer
0	QC4-01	32.2	30.7	95.2	95.3	48.8	42.1	86.5	89.8
0	QC4-02	32.2	30.7	95.3	(0.0)	48.8	45.4	93.1	(5.2)
5	QC4-03	32.2	31.1	96.4	94.2	48.8	45.6	93.5	90.7
5	QC4-04	32.2	29.6	92.0	(3.3)	48.8	42.8	87.8	(4.5)
	pH 7 buffer
0	QC7-01	32.2	29.4	91.3	93.4	48.8	43.9	90.0	91.6
0	QC7-02	32.2	30.8	95.5	(3.2)	48.8	45.4	93.2	(2.5)
5	QC7-03	32.2	30.1	93.2	92.2	48.8	44.5	91.4	88.0
5	QC7-04	32.2	29.4	91.1	(1.7)	48.8	41.3	84.7	(5.4)
	pH 9 buffer
0	QC9-01	32.2	22.2	68.8	69.8	48.8	49.8	102.1	107.7
0	QC9-02	32.2	22.8	70.9	(2.1)	48.8	55.2	113.3	(7.4)
5	QC9-03	32.2	26.4	81.9	82.3	48.8	47.7	97.9	100.8
5	QC9-04	32.2	26.7	82.7	(0.7)	48.8	50.5	103.7	(4.1)

 

Table 4: Concentrations and recovery of 2,2-Difluoroethyl acetate and 2,2-difluoroethanol (DFE) in aqueous pH 9.0 buffered solutions analyzed by HS/GC/MS (Tier 1)

Sampling Interval (days)	Sample ID (112E-142-)	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Mean (CV)	Percent Dose (%)	Analyzed Conc. (nM/mL)	Mean (CV)	Percent Dose (%)
0	17	32.7	18.3	13.6	41.8	8.1	6.8	21.1	20.4	63.0
	18	32.2	13.2	(33.7)		5.6	(25.7)
	19	32.2	9.2			<LOQ ^1
5	20	32.2	0.7	0.69	2.1	32.6	31.0	95.8	31.7	97.9
	21	32.2	0.68	(1.4)		30.1	(4.4)
	22	32.7	0.68			30.3

1 LOQ: limit of quantitation.

 

Table 5: Concentrations and recovery of 2,2-Difluoroethyl acetate and 2,2-difluoroethanol (DFE) in aqueous pH 7.0 buffered solutions analyzed by HS/GC/MS (Tier 1)

Sampling Interval (days)	Sample ID (112E-142-)	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Mean (CV)	Percent Dose (%)	Analyzed Conc. (nM/mL)	Mean (CV)	Percent Dose (%)
0	9	32.2	31.8	28.0	86.8	<LOQ ^1	NA ^2	NA	28.0	86.8
	10	32.2	15.0	(41.2)		<LOQ	NA
	11	32.2	37.1			<LOQ
5	12	32.2	6.0	5.3	16.6	22.5	24.2	75.6	29.6	92.2
	13	31.8	5.1	(10.4)		25.5	(6.4)
	14	32.2	4.9			24.7

1 LOQ: limit of quantitation.
2 NA: not applicable.

 

Table 6: Concentrations and recovery of 2,2-Difluoroethyl acetate and 2,2-difluoroethanol (DFE) in aqueous pH 4.0 buffered solutions analyzed by HS/GC/MS (Tier 1)

Sampling Interval (days)	Sample ID (112E-142-)	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Mean (CV)	Percent Dose (%)	Analyzed Conc. (nM/mL)	Mean (CV)	Percent Dose (%)
0	1	32.2	19.8	17.5	54.6	<LOQ ^1	NA	NA	17.5	54.6
	2	32.2	14.6	(15.2)		<LOQ	NA
	3	31.8	18.2			<LOQ
5	4	32.2	28.5	27.3	84.6	1.2	1.1	3.4	28.4	88.0
	5	32.2	25.3	(6.2)		0.97	(11.1)
	6	32.2	27.9			1.15

1 LOQ: limit of quantitation.
2 NA: not applicable.

 

Table 7: Concentrations and recovery of 2,2-Difluoroethyl acetate and 2,2-difluoroethanol (DFE) quality control samples in aqueous pH 9.0 buffered solutions analyzed by HS/GC/MS (Tier 2)

Sampling Interval (days)	Sample ID	2,2-Difluoroethyl acetate				DFE
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0	112E-142QC9-1	32.2	28.7	89.0	87.1	48.8	48.1	98.7	97.1
0	112E-142QC9-2	32.2	27.5	85.2		48.8	46.5	95.4
1	112E-142QC9-3	32.2	27.1	84.2	88.5	48.8	43.6	89.5	94.8
1	112E-142QC9-4	32.2	29.9	92.7		48.8	48.8	100.2
2	112E-142QC9-5	32.2	26.0	80.6	82.3	48.8	43.8	89.8	90.1
2	112E-142QC9-6	32.2	27.1	84.0		48.8	44.1	90.4
3	112E-142QC9-7	32.2	26.3	81.7	84.9	48.8	41.5	85.2	89.1
3	112E-142QC9-8	32.2	28.4	88.1		48.8	45.4	93.0
7	112E-142QC9-9	32.2	27.1	84.1	85.9	48.8	43.0	88.2	91.6
7	112E-142QC9-10	32.2	28.3	87.8		48.8	46.3	95.0

 

 

Table 8: Concentrations and recovery of 2,2-Difluoroethyl acetate and 2,2-difluoroethanol (DFE) quality control samples in aqueous pH 7.0 buffered solutions analyzed by HS/GC/MS (Tier 2)

Sampling Interval (days)	Sample ID	2,2-Difluoroethyl acetate				DFE
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0	112E-142QC7-1	32.2	30.7	95.3	95.5	48.8	45.2	92.7	95.6
0	112E-142QC7-2	32.2	30.9	95.8		48.8	48.0	98.5
1	112E-142QC7-3	32.2	29.0	89.8	91.0	48.8	40.3	82.6	85.6
1	112E-142QC7-4	32.2	29.7	92.1		48.8	43.2	88.7
2	112E-142QC7-5	32.2	28.2	87.3	90.0	48.8	45.6	93.6	95.3
2	112E-142QC7-6	32.2	29.9	92.7		48.8	47.3	96.9
3	112E-142QC7-7r*	32.2	28.7	89.0	86.5	48.8	43.9	90.1	87.5
3	112E-142QC7-8	32.2	27.0	83.9		48.8	41.3	84.8
7	112E-142QC7-9	32.2	28.9	89.7	87.1	48.8	44.9	92.1	88.8
7	112E-142QC7-10	32.2	27.2	84.4		48.8	41.7	85.4
14	112E-142QC7-11	32.2	25.3	78.5	82.1	48.8	40.7	83.6	85.3
14	112E-142QC7-12	32.2	27.6	85.7		48.8	42.5	87.1
21	112E-142QC7-13	32.2	26.8	83.1	85.6	48.8	48.9	100.3	102.4
21	112E-142QC7-14	32.2	28.4	88.1		48.8	50.9	104.5
32	112E-142QC7-15r*	32.2	32.7	101.3	102.4	48.8	48.1	98.7	101.8
32	112E-142QC7-16	32.2	33.4	103.5		48.8	51.1	104.9

* = Injection repeated due to unacceptable initial injection.

 

 

Table 9: Tier 2: 2,2-Difluoroethyl acetate hydrolysis summary – pH 9 at 20°C

Sampling Interval (days)	Sample ID 112E-142A-	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0.00	1r*	32.6	30.6	94.1	96.6	2.8	8.6	10.7	102.6	107.3
0.00	2r*	32.1	31.9	99.2		4.1	12.9		112.1
0.5	4	32.2	23.7	73.7	69.4	8.8	27.3	31.1	100.9	100.5
0.5	5r*	32.3	21.1	65.2		11.2	34.8		100.1
1	6	32.0	16.6	52.0	50.3	11.8	37.0	35.2	89.0	85.5
1	7	32.4	15.8	48.7		10.8	33.4		82.1
2	8	32.5	10.6	32.7	32.9	19.5	59.9	61.5	92.6	94.4
2	9	32.0	10.6	33.1		20.2	63.0		96.2
3	10	32.6	7.2	22.2	21.7	25.2	77.1	75.7	99.3	97.4
3	11	32.0	6.8	21.3		23.7	74.3		95.5
7	12	31.9	1.4	4.5	4.2	30.1	94.3	92.4	98.8	96.6
7	13	32.5	1.3	4.0		29.4	90.5		94.5

* = Injection repeated due to unacceptable initial injection.

 

 

Table 10: Tier 2: 2,2-Difluoroethyl acetate hydrolysis summary– pH 9 at 35°C

Sampling Interval (days)	Sample ID 112E-142A-	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0.0	1r*	32.6	30.6	94.1	96.6	2.8	8.6	10.7	102.6	107.3
0.0	2r*	32.1	31.9	99.2		4.1	12.9		112.1
0.125	17	32.0	20.6	64.5	61.9	10.3	32.0	31.6	96.6	93.6
0.125	18	32.6	19.3	59.3		10.2	31.2		90.6
0.25	19	32.0	14.5	45.3	42.7	19.4	60.7	62.2	106.1	104.9
0.25	20r*	32.3	12.9	40.0		20.6	63.7		103.7
0.5	21	32.5	8.6	26.3	26.7	24.9	76.7	80.7	103.1	107.5
0.5	22	32.5	8.8	27.2		27.6	84.7		111.9
1	23	32.4	2.3	7.2	7.6	28.0	86.6	88.4	93.7	96.0
1	24	32.3	2.6	8.0		29.2	90.3		98.3
2	25	32.5	0.5	1.4	1.4	31.0	95.5	96.6	96.9	97.9
2	26	31.9	0.4	1.3		31.2	97.6		98.9

* = Injection repeated due to unacceptable initial injection.

 

 

Table 11: Tier 2: 2,2-Difluoroethyl acetate hydrolysis summary – pH 9 at 50°C

Sampling Interval (days)	Sample ID 112E-142A-	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0.0	1r*	32.6	30.6	94.1	96.6	2.8	8.6	10.7	102.6	107.3
0.0	2r*	32.1	31.9	99.2		4.1	12.9		112.1
0.0625	30	32.1	21.3	66.4	69.7	11.9	36.9	36.4	103.3	106.1
0.0625	31	32.3	23.6	73.1		11.6	35.8		108.9
0.125	32	32.4	10.0	30.7	30.0	24.9	76.8	76.5	107.5	106.5
0.125	33	32.2	9.5	29.3		24.6	76.3		105.6
0.25	34	32.3	1.4	4.4	4.3	29.2	90.5	87.4	94.9	91.6
0.25	35	32.7	1.3	4.1		27.6	84.3		88.4
0.5	36r*	32.3	0.4	1.3	1.3	26.7	82.6	85.4	83.8	86.7
0.5	37	32.0	0.4	1.2		28.3	88.3		89.5
1	38	32.3	0.3	0.9	0.9	29.0	89.9	89.9	90.8	90.7
1	39	32.1	0.3	0.9		28.8	89.8		90.7

* = Injection repeated due to unacceptable initial injection.

 

 

Table 12: Tier 2: 2,2-Difluoroethyl acetate hydrolysis summary – pH 7 at 20°C

Sampling Interval (days)	Sample ID 112E-142A-	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0	43r*	31.8	35.7	112.4	114.5	0.0	NA^1	NA^1	112.4	114.8
0	44r*	31.9	37.2	116.7		0.2	0.5		117.2
3	46	32.5	26.8	82.5	84.6	1.1	3.4	3.4	85.9	88.0
3	47	32.2	27.9	86.7		1.1	3.4		90.1
7	48	31.8	25.0	78.4	81.7	2.3	7.2	6.0	85.7	87.6
7	49	32.5	27.5	84.9		1.5	4.7		89.6
14	50	32.1	24.8	77.3	75.1	3.4	10.5	11.3	87.8	86.4
14	51	32.3	23.5	72.8		3.9	12.1		85.0
21	52	32.4	23.7	73.3	72.2	5.7	17.7	15.3	91.0	87.5
21	53	32.0	22.8	71.2		4.1	12.8		84.0
32	54	32.1	19.6	60.9	62.8	6.6	20.4	19.6	81.4	82.4
32	55	32.2	20.8	64.6		6.0	18.8		83.4

1 NA = Not Applicable.
* = Injection repeated due to unacceptable initial injection.

 

 

Table 13: Tier 2: 2,2-Difluoroethyl acetate hydrolysis summary – pH 7 at 35 °C

Sampling Interval (days)	Sample ID 112E-142A-	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0	43r*	31.8	35.7	112.4	114.5	0.0	NA^1	NA^1	112.4	114.8
0	44r*	31.9	37.2	116.7		0.2	0.5		117.2
3	59	32.2	23.5	72.9	72.8	4.0	12.6	12.8	85.5	85.5
3	60	32.4	23.5	72.6		4.2	12.9		85.6
7	61	32.2	19.5	60.5	59.9	8.7	27.0	27.0	87.6	86.8
7	62	31.9	18.9	59.2		8.6	26.9		86.1
14	63	32.1	13.4	41.7	41.8	15.1	47.0	46.3	88.7	88.1
14	64	31.8	13.3	41.9		14.5	45.6		87.4
21	65	32.2	8.5	26.4	25.1	23.2	72.1	70.9	98.5	96.1
21	66	32.1	7.7	23.8		22.4	69.7		93.6
32	67	32.1	3.8	11.8	11.7	24.5	76.4	73.7	88.1	85.4
32	68	32.0	3.7	11.6		22.7	71.0		82.6

1 NA = Not Applicable.
* = Injection repeated due to unacceptable initial injection.

 

 

Table 14: Tier 2: 2,2-Difluoroethyl acetate hydrolysis summary– pH 7 at 50 °C

Sampling Interval (days)	Sample ID 112E-142A-	2,2-Difluoroethyl acetate				DFE			Total Recovery (nM/mL)	Total Recovery (% dose)
		Dose Conc. (nM/mL)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)	Analyzed Conc. (nM/mL)	Percent Dose (%)	Mean (%)
0	43r*	31.8	35.7	112.4	114.5	0.0	NA^1	NA^1	112.4	114.8
0	44r*	31.9	37.2	116.7		0.2	0.5		117.2
1	72	32.1	21.8	67.9	68.4	6.5	20.2	20.9	88.1	89.3
1	73	32.1	22.1	68.9		6.9	21.6		90.5
2	74	32.0	19.4	60.6	58.8	11.4	35.7	35.9	96.3	94.7
2	75	32.2	18.4	57.1		11.6	36.1		93.2
3	76	32.2	13.8	42.9	42.0	14.5	45.0	46.3	87.8	88.3
3	77	32.1	13.2	41.2		15.3	47.6		88.7
7	78	32.2	4.6	14.4	13.9	25.1	77.8	78.2	92.3	92.1
7	79	31.9	4.3	13.4		25.1	78.5		91.9
14	80	32.4	0.4	1.4	1.0	26.2	80.8	79.4	82.2	80.3
14	81	32.0	0.2	0.5		25.0	77.9		78.5

1 NA = Not Applicable.
* = Injection repeated due to unacceptable initial injection.

 

 

Table 15: Tier 2: 2,2-Difluoroethyl acetate linear regression summary – pH 9

20 °C		35 °C		50 °C
Time	Ln(C0/Ct)	Time	Ln(C0/Ct)	Time	Ln(C0/Ct)
0.00	-0.0613	0.00	-0.0613	0.00	-0.0613
0.00	-0.008	0.00	-0.008	0.00	-0.008
0.5	-0.3058	0.125	-0.4383	0.0625	-0.4097
0.5	-0.4271	0.125	-0.5222	0.0625	-0.3137
1.00	-0.6545	0.25	-0.7917	0.125	-1.1807
1.00	-0.7193	0.25	-0.9162	0.125	-1.2259
2.00	-1.1172	0.5	-1.3345	0.25	-3.1189
2.00	-1.1051	0.5	-1.3034	0.25	-3.188
3.00	-1.5054	1.00	-2.6363	0.5	-4.3593
3.00	-1.5473	1.00	-2.5305	0.5	-4.3839
7.00	-3.106	2.00	-4.2718	1.00	-4.7474
7.00	-3.2305	2.00	-4.3349	1.00	-4.7321
Slope	-0.4361	Slope	-2.4635	Slope	-12.917
Intercept	-0.1676	Intercept	-0.1304	Intercept	0.2245
r2	0.9928	r2	0.9911	r2	0.9698

 

 

Table 16: Tier 2: 2,2-Difluoroethyl acetate linear regression summary – pH 7

20 °C		35 °C		50 °C
Time	Ln(C0/Ct)	Time	Ln(C0/Ct)	Time	Ln(C0/Ct)
0	0.1166	0	0.1166	0	0.1166
0	0.1541	0	0.1541	0	0.1541
3	-0.1927	3	-0.3154	1	-0.3865
3	-0.1428	3	-0.32	1	-0.3722
7	-0.2427	7	-0.5018	2	-0.5011
7	-0.1639	7	-0.5242	2	-0.5605
14	-0.2577	14	-0.8741	3	-0.8472
14	-0.3169	14	-0.8701	3	-0.8873
21	-0.3112	21	-1.3301	7	-1.9355
21	-0.3393	21	-1.4333	7	-2.0091
32	-0.4955	32	-2.1408	14	-4.2982
32	-0.4367	32	-2.1564	14	-5.215
Slope	-0.0148	Slope	-0.0672	Slope	-0.3402
Intercept	-0.0289	Intercept	0.0126	Intercept	0.1356
r2	0.764	r2	0.9876	r2	0.9793

 

 

Table 17: Arrhenius equation for the estimation of the 25 °C DT50 for 2,2-Difluoroethyl acetate incubated at pH 9 and 7

Buffers	Temperature (°C)	1/T (temperature. 1/K)	lnk	Linear regression analysis (slope and y-intercept)		25°C
						ln k'	k'	DT50 (days)
pH 9	20	0.0034	-0.8298	Slope -Ea/R =	-10694.84	-0.232	0.7927	0.87
	35	0.0032	0.9016	Intercept ln(A) =	35.64
	50	0.0031	2.5586	Ea =	88.92
pH 7	20	0.0034	-4.2121	Slope -Ea/R =	-9882.2	-3.688	0.025	27.7
	35	0.0032	-2.7003	Intercept ln(A) =	29.46
	50	0.0031	-1.0783	Ea =	82.16

Arrhenius equation Ln k = -Ea/RT + Ln (A)
k = rate constant, measured at different temperatures
Ea = activation energy (KJ/mol)
T = absolute temperature [K] = 273.15 + T (Celsius °C)
R = gas constant [8.314J/mol*K]
k' = rate constant, predicted at 25 °C using Arrhenius equation
* Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

Validity criteria fulfilled:

not applicable

Remarks:

According to the OECD 111 guideline, there is no validity criteria but quality criteria. The validity criteria for recovery, repeatability and sensibility of the analytical method were fulfilled.

Conclusions:

Hydrolysis is expected to contribute to the degradation of 2,2-Difluoroethyl acetate at pH 7.0 and pH 9.0, but not at pH = 4 at which 2,2-Difluoroethyl acetate was hydrolytically stable. The half-life at 25°C was calculated to be 28 days at pH 7.0, 0.9 days at pH 9.0 and greater than one year at pH 4.0.

Executive summary:

The hydrolysis behaviour of 2,2-Difluoroethyl acetate was investigated in a study performed according to OECD test guideline 111 under GLP compliance, and conducted in two subsequent tiers.

In tier 1, DFEA solutions with a concentration of ca. 4 mg/L were prepared in buffered solutions at pH = 4, 7 and 9 and incubated in the dark for five days at 50 °C. The concentrations of DFEA and of the transformation product 2,2-difluoroethanol were measured in triplicates at test initiation and termination using headspace gas chromatography mass spectrometry (HS/GC/MS).

During tier 1, 3.9, 83 and 98 % degradation were observed at pH = 4, 7 and 9, respectively. Based on the mass balance analysis on Day 5 and on the knowledge of DFEA hydrolysis mechanisms, 2,2-difluoroethanol was considered to be the principal hydrolytic degradation product. pH = 7 and 9 were selected for further investigations in tier 2.

In tier 2, DFEA solutions with a concentration of ca. 4 mg/L were thus prepared in buffered solutions at pH = 7 and 9 and incubated in the dark at 20, 35 and 50 °C. The concentrations of DFEA and of the transformation product 2,2-difluoroethanol were measured in duplicates at various sampling intervals using HS/GC/MS:

- pH = 7:

→ 20 and 35 °C: after 3, 7, 14, 21 and 32 days.

→ 50 °C: after 1, 2, 3, 7 and 14 days.

- pH = 9

→ 20 °C: after 0.5, 1, 2, 3 and 7 days.

→ 35 °C: after 0.125, 0.25, 0.5, 1 and 2 days.

→ 50 °C: after 0.0625, 0.125, 0.25, 0.5 and 1 day.

During tier 2, at pH = 7, the average initial amount of DFEA (115 % on day 0) decreased to 63, 12 and 1 % at 20 °C (day 32), 35 °C (day 32) and 50 °C (day 14), respectively. At this pH, the average amount of the transformation product, 2,2-difluoroethanol, increased from below the limit of quantification on day 0 to 20, 74 and 79 % at 20 °C (day 32), 35 °C (day 32) and 50 °C (day 14), respectively. At pH = 9, the average initial amount of DFEA (97 % on day 0) decreased to 4.2, 1.4 and 1 % at 20 °C (day 7), 35 °C (day 2) and 50 °C (day 1), respectively. At this pH, the average amount of the 2,2-difluoroethanol increased from below 11 % on day 0 to 92, 97 and 90 % at 20 °C (day 7), 35 °C (day 2) and 50 °C (day 1), respectively.

From the two tiers, it is concluded that hydrolysis is expected to contribute to the degradation of DFEA at pH = 7 and 9, but not at pH = 4 at which DFEA was hydrolytically stable. The half-lives at 25 °C were calculated to be greater than one year, 28 days and 0.9 days at pH = 4, 7 and 9, respectively.

Description of key information

Hydrolysis is expected to contribute to the degradation of 2,2-Difluoroethyl acetate at pH 7.0 and pH 9.0, but not at pH = 4 at which 2,2-Difluoroethyl acetate was hydrolytically stable. The half-life at 25°C was calculated to be 28 days at pH 7.0, 0.9 days at pH 9.0 and greater than one year at pH 4.0.

Key value for chemical safety assessment

Half-life for hydrolysis:

28 d

at the temperature of:

25 °C

Additional information

An experimental study performed under GLP compliance and in accordance with OECD test guideline 111 was flagged as a key study and assigned a Klimisch score of 1. It reports that the half-life of 2,2-Difluoroethyl acetate at 25 ºC was calculated to be 28 days at pH 7.0 and 0.9 days at pH 9.0. 2,2-Difluoroethyl acetate is considered as hydrolytically stable at pH 4.0 with a nominal half-life of greater than one year.