Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Feb 1993 to 12 Aug 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals Duplicate test solution vials were removed from the incubator and analysed at the following time intervals: 0, 1, 3, 7. 10, 15, 20, 24 and 30 days for pH 5 and 7; 0, 1, 3, 7, 10, 17, 20, 24 and 30 days for pH 9. At the time of sampling, the appropriate sampling interval and replicate number were added to each sample label.
- Sample storage: All collected test solutions were stored from February 1, 1993 to March 2, 1993 in a freezer that was maintained at < -20°C. After March 2, 1993, all collected test solutions vials were stored in a refrigerator maintained at 0 5°C. The storage of test solutions were changed because some test solutions that were frozen after collection and analysis had low (29.2% to 106.9%) accountability of the 14C material upon defrosting, equilibrating to room temperature, and reanalysis of selected test solutions. Test solutions that were stored in the refrigerator after collection and analysis, maintained acceptable (100 ± 10%) accountability of 14C material upon reanalysis of selected test solutions after equilibrating the test solutions to room temperature. Attempts were made to resolubilize the 14C material in the test solutions that were frozen with organic solvents. lsopropanol added at 100µL increments up to approximately 15% by volume of the test solution followed by the addition of acetone to the same test solution failed to resolubilize the 14C material. After each manipulation of the test solution, the test solution was vortexed and sonicated to thoroughly mix the test solution.
All reference substances and solutions of reference substance were stored in a refrigerator maintained at 0 ± 5°C. The 14C-labelled test substance was stored in a freezer maintained at < - 20°C, and solutions of the test substance were stored in a refrigerator maintained at 0.5°C. Solutions of the test and reference substances were protected from light by covering them with aluminium foil. The test substance was protected from light by using a low actinic vial, and the reference substances were protected from light by keeping them in an opaque plastic container.

Buffers:

- pH 5: 0.01 M Acetic Acid - Sodium Acetate - 884.5 mg of sodium acetate trihydrate (CH3COONa•3H2O) was added to 200 μL of glacial acetic acid (CH3COOH) in 300 mL of water. The pH was adjusted to pH 5.0 ± 0.01 with 0.1 M NaOH then water was added to a final volume of one litre.
- pH 7: 0.01 M Sodium Dihydrogen Phosphate Disodium Hydrogen Phosphate - 539.4 mg of disodium hydrogen phosphate Na2HPO4) and 855.5 mg of sodium dihydrogen phosphate hydrate (NaH2PO4•H2O) was dissolved in approximately 300 mL of water. The pH was adjusted to pH 7.0 ± 0.01 with 0.1 M NaOH and 0.1 M phosphoric acid then water was added to a final volume of one litre.
- pH 9: 0.01 M Sodium Borate - Hydrochloric Acid - 3.8140 g of sodium borate decahydrate (Na2B4O7·10 H20) was added to approximately 300 mL of water. pH was adjusted to pH 9.0 ± 0.01 with 0.1 M hydrochloric acid then water was added to a final volume of one litre.

Prior to use, each buffer solution was sterilized by filtration through a Nalgene filter apparatus equipped with a 0.2 micron cellulose nitrate filter membrane.

Details on test conditions:

TEST SYSTEM
- Test vials: Each test vial, except the bulk dose vial, consisted of a 3 mL amber borosilicate glass container with a silicone septum and an open-top plastic screw cap. Each bulk dose vial consisted of a 30 mL amber borosilicate glass container with a silicone septum and an open-top plastic screw cap.
- Silylation of glassware: Silylated glassware (All glassware used throughout this study was silylated. The silylating solution contained 5% dichlorodimethylsilane and 95% methylene chloride The glassware was soaked in the solution at least 15 minutes at room temperature, then emptied, and rinsed with copious amounts of methanol (MeOH). The glassware was given a final rinse with methylene chloride, allowed to air dry, and heated in an oven at 110 °C for a minimum of two hours.)
- Sterilisation method: Except for glassware that was purchased pre-sterilized, all glassware, including test system and bulk dose vials, septa, were sterilized before use by autoclaving for approximately 20 minutes at 15 psig and 121°C.
- Incubation: All dosed test solution vials were wrapped with aluminum foil, placed in an aluminum wrapped box. then placed in a constant temperature incubator. The samples were incubated at 25.1°C, under dark conditions, for up to 30 days.
- Temperature: pH 5: 24.9 ± 0.16 ˚C; pH 7: 24.9 ± 0.15 ˚C; pH 9: 25.0 ± 0.21 ˚C
- Temperature monitoring: The temperature of the incubator was periodically monitored by reading the incubator's calibrated temperature LED readout and manually recording the temperature. In addition to the manual observation and recordings, the temperature was continuously monitored by using a recording thermometer.
- pH measurement: The pH of each test solution was measured and recorded after dosing (0 Day) and at each subsequent sampling point.

Duration:

30 d

Temp.:

25 °C

Initial conc. measured:

2.1 mg/L

Remarks:

applicable to all pH values

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Test performance:

- Test solution sterility: The test solutions at all pH’s evaluated were considered sterile during the duration of the study. Virtually no colony growth was observed for any of the aliquots after at least three days of incubation on agar medium at 25 ±1°C.
Aliquots of the test solutions were plated at each sampling interval. The test solutions for pH 9 buffer, 15 day interval, appeared to be non-sterile because 3 to 4 colony forming units grew on each of three agar plates. This interval was not used to generate any data presented in this report; duplicate extra test vials were collected at day 17 to replace the day 15 interval.

Transformation products:

no

% Recovery:

> 100

pH:

5

Temp.:

25 °C

Duration:

30 d

% Recovery:

> 100

pH:

7

Temp.:

25 °C

Duration:

30 d

% Recovery:

> 100

pH:

9

Temp.:

25 °C

Duration:

30 d

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 d-1

DT50:

3 136 d

Type:

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

pH:

5

Temp.:

25 °C

Hydrolysis rate constant:

0 d-1

DT50:

1 406 d

Type:

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

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

-0 d-1

DT50:

4 101 d

Type:

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

Details on results:

An overview of the results is provided in Table 1 - Table 4 in 'Any other information on results incl. tables'.
- pH conditions maintained throughout the study: Yes; The test solutions measured 5.00 ± 0.06 (range 4.92 to 5.13), 7.02 ± 0.06 (range 6.93 to 7.12), and 9.08 ± 0.03 (range 9.03 to 9.14) for pH 5, 7 and 9 test solutions, respectively.
- Sterility condition maintained throughout the study: Yes; The test solutions at all pH’s evaluated were considered sterile during the duration of the study. Virtually no colony growth was observed for any of the aliquots after at least three days of incubation on agar medium at 25 ±1°C. Aliquots of the test solutions were plated at each sampling interval. The test solutions for pH 9 buffer, 15 day interval, appeared to be non-sterile because 3 to 4 colony forming units grew on each of three agar plates. This interval was not used to generate any data presented in this report; duplicate extra test vials were collected at day 17 to replace the day 15 interval.

- Material balance: Material balances were calculated based on the dose solution (n = 5) determined on 0 Day for each pH buffered solution. The dose concentration for pH 5, 7, and 9 was 2.10 ppm, 2.10 ppm, and 2.04 ppm, respectively. The material balances were 101.1 ± 1.9 (range 98.0 to 104.5), 1004 ± 1.4 (range 98.0 to 103.7), and 104.2 ± 2.2 (range 99.5 to 109.3) percent of the applied radioactivity for pH 5, 7 and 9, respectively.

- Characterization: For all pH's and all sampling intervals, the test substance accounted for greater than 95% (normalized) of the applied dose. The mean HPLC column recoveries were 101.2 ± 3.7%, 102.6 ± 5.3% and 102.5 ± 3.1% for pH 5, 7 and 9, respectively. The limit of detection (LCD) for these experiments ranged from 0.012 to 0.018 ppm.
Quantitation of the parent and its metabolites in the test solutions was based on DPM's assayed from the scraped sections of the TLC plate for each test solution as determined by LSC. For all pH's and all sampling intervals tested, the test substance accounted for greater than or equal to 95% (normalized) of the applied dose at 0 Day. The mean TLC plate recoveries were 91.3 ± 4.1%, 90.8 ± 3.4% and 90.9 ± 4.2%. The limit of detection (LCD) for these experiments ranged from 0.00088 to 0.0034 µg.

- Isolation of degradates for characterization: There were no degradates of the test substance (≥ 10% of the applied radioactivity) formed under pH 5, 7 or 9 aqueous buffered conditions. One possible minor degradate was detected in the 13 minute HPLC eluant of several test solutions. The maximum concentration of the 13 minute HPLC unknown was 6.24% the pH 9,10 Day Rep. 1 test solution.
Test solutions containing the 13 minute HPLC eluant unknown (pH 5 - 30 Day Rep. 1; pH 7 - 20 Day Rep. 1 and 2, 24 Day Rep. 1 and 2; and pH 9 0 Day Rep. 2, 7 Day Rep. 1 and 2, 10 Day Rep. 1 and 2) were pooled, thoroughly mixed, separated by HPLC, fractions collected, and subsequently analysed by LSC. The 13 minute HPLC eluant unknown was not detected in the pooled sample. Three test solutions (pH 9 - 0 Day Rep. 1, 7 Day Rep. 1, and 15 Day Rep. 2) that contained the 13 minute HPLC eluant unknown but were not pooled, were reanalysed by HPLC. Upon reanalysis, only the pH 9 - 0 Day Rep. 1 contained the 13 minute HPLC eluant unknown; test solutions pH 9 - 7 Day Rep. 1 and 15 Day Rep. 2 did not contain the 13 minute HPLC eluant unknown. The pooled sample was concentrated 20 fold to ensure the disappearance of the 13 minute HPLC eluant unknown was not due to dilution. Although the test substance was not soluble at this concentration, the aqueous layer was separated by HPLC, fractions collected and subsequently analysed by LSC. The 13 minute HPLC eluant unknown was not present. The test substance remaining in the test tube was dissolved in hexane then the hexane was exchanged to acetonitrile. The acetonitrile solution separated by HPLC, fractions collected, and subsequently analysed by LSC. The 13 minute HPLC eluant unknown was not present. Since the appearance of the 13 minute HPLC eluant unknown was sporadic, and the availability of test solutions containing the degradate was diminished, no further attempts to identify the 13 minute degradate were possible. The 13 minute HPLC eluant unknown was not detected in any of the bulk dose test solutions. There were no degradates greater than 5% of the applied dose (pH7 – 20 Day Rep. 2) observed in the test solutions analysed by TLC.
The erratic occurrence of the 13 minute HPLC eluant unknown, and its detection in both replicates of the 0 day pH 9 test solutions, suggest that this unknown is not a hydrolytic degradate of the test substance. The author postulates that the 13 minute HPLC eluant unknown was a contaminant, or impurity, that was transient and possibly adhered to glassware.

- Half-life: The calculated half-life values for the test substance were 1406 days, 3136 days and 4101 days under pH 5, 7, and 9 conditions, respectively.

Table 1. HPLC Column Recoveries Under pH 5, 7 and 9 Conditions

	Percent recovery
Interval (Days)	replicate	pH 5	pH 7	pH 9
0	1	93.7	98.6	102.2
	2	99.8	99.6	101.5
1	1	103.4	101.9	104.6
	2	98.0	101.5	100.0
3	1	101.2	98.7	96.2
	2	100.4	93.7	96.2
7	1	105.3	100.6	107.8
	2	100.1	98.2	102.9
10	1	96.4	113.0	102.7
	2	101.6	104.8	101.8
15	1	106.0	97.1	NA
	2	101.5	98.6	NA
17	1	NA	NA	106.6
	2	NA	NA	102.1
20	1	103.0	105.9	103.8
	2	97.7	102.7	103.3
24	1	109.9	110.0	103.7
	2	102.3	109.7	105.2
30	1	101.2	110.3	105.1
	2	100.7	102.6	100.0
Mean	=	101.2	102.6	102.5
Standard deviation	=	3.7	5.3	3.1
n	=	18	18	18

 NA: not analyzed

 

Table 2. Half-life of the test substance based on HPLC analysis of pH 5 test solutions

Sampling days	Average Normalized Day Percent of Total Radioactivity	In of average Normalized Percent of Total Radioactivity as Parent
0	99.10	4.596
1	99.48	4.600
3	99.66	4.602
7	100.00	4.605
10	99.22	4.597
15	98.67	4.592
20	99.12	4.596
24	99.14	4.597
30	97.44	4.597

Correlation coefficient = 0.7106

Intercept (1/n) = 4.6020

Slope (-k) = 4.93E-04

T1/2 = 1406 days

 

Table 3. Half-life of the test substance based on HPLC analysis of pH 7 test solutions

Sampling days	Average Normalized Day Percent of Total Radioactivity	In of average Normalized Percent of Total Radioactivity as Parent
0	100.00	4.605
1	99.02	4.595
3	99.49	4.600
7	98.74	4.592
10	98.70	4.592
15	99.10	4.596
20	98.80	4.593
24	97.78	4.583
30	99.60	4.601

Correlation coefficient = 0.3530

Intercept (1/n) = 4.5980

Slope (-k) = 2.21E-04

T1/2 = 3136 days

 

Table 4. Half-life of the test substance based on HPLC analysis of pH 9 test solutions

Sampling days	Average Normalized Day Percent of Total Radioactivity	In of average Normalized Percent of Total Radioactivity as Parent
0	97.74	4.582
1	99.64	4.602
3	99.16	4.597
7	97.56	4.580
10	95.44	4.558
15	99.26	4.598
20	98.78	4.593
24	99.10	4.596
30	98.54	4.590

Correlation coefficient = 0.13453

Intercept (1/n) = 4.5863

Slope (-k) = -1.69E-04

T1/2 = 4101 days

Validity criteria fulfilled:

yes

Conclusions:

In a hydrolysis study performed in accordance with EPA 161-1 and under GLP, the calculated half-lives of the test substance using pseudo-first order reaction kinetics were 1406 days, 3136 days and 4101 days for pH 5, 7 and 9 conditions, respectively. Thus, it was conclude that the test substance is hydrolytically stable under acidic, neutral. and basic conditions. Based on these data, hydrolysis would not be expected to be a major degradative pathway for the test substance.

Executive summary:

The hydrolysis of the substance was studied according to the EPA 161-1 guideline and in compliance with GLP criteria. Φ(B)14C- labelled substance was prepared in acetonitrile and applied to sterile pH 4, 5, 7 and 9 aqueous buffer solutions for final concentrations of 2.10 ppm, 2.10 ppm and 2.04 ppm, respectively. The test solution vials were wrapped in aluminum foil and were incubated in dark at 25 ± 1˚C for up to 30 days. Duplicate pH 5 and 7 test solution vials were sampled at 0, 1, 3, 7, 10, 15, 20, 24 and 30 days. Duplicate pH 9 test solution vials were sampled at 0, 1. 7, 10, 17, 20, 24 and 30 days. With the exception of 0 day pH 5, both test solution vials were used for liquid scintillation counting (material balance), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and pH measurement. Sterility measurements were conducted on only one of the test solution vials for each sampling interval. The test solutions maintained sterility and the pH was constant throughout the study.

The parent concentrations were essentially constant under pH 5, 7 and 9 conditions for the entire 30 day study period. No major degradate (10% of applied radioactivity) was formed under pH 5, 7 and 9 conditions. The material balance was 101.1 ± 1.9% under pH 5, 100.4 ± 1.4% under pH 7, and 104.2 ± 2.2% under pH 9 conditions. The calculated half-lives of the test substance using pseudo-first order reaction kinetics were 1406 days, 3136 days and 4101 days for pH 5, 7 and 9 conditions, respectively. The calculated rate constants of the test substance were 4.97E-04/day, 2.21E-04/day and -1.69E-04/day for pH 5, 7 and 9 conditions, respectively. The half-lives and the rate constants were calculated using HPLC data. The test substance has been shown to be hydrolytically stable under acidic, neutral, and basic conditions. The DT50 values were calculated to be 1406, 3136 and 4101 days in pH 5, pH 7 and pH 9 conditions, respectively. Based on these data, hydrolysis would not be expected to be the major degradative pathway for the test substance.

Description of key information

All available data was assessed and the studies representing the worst-case effects were included as key study. Other studies are included as supporting information.

Hydrolytic DT50 at pH 7 at 25 °C = 3136 days, hydrolytically stable under acidic, neutral, and basic conditions, EPA 161 -1, Thomas 1994

Key value for chemical safety assessment

Half-life for hydrolysis:

3 136 d

at the temperature of:

25 °C

Additional information

The hydrolysis of the substance was studied according to the EPA 161-1 guideline and in compliance with GLP criteria. Φ(B)14C- labelled substance was prepared in acetonitrile and applied to sterile pH 4, 5, 7 and 9 aqueous buffer solutions for final concentrations of 2.10 ppm, 2.10 ppm and 2.04 ppm, respectively. The test solution vials were wrapped in aluminum foil and were incubated in dark at 25 ± 1˚C for up to 30 days. Duplicate pH 5 and 7 test solution vials were sampled at 0, 1, 3, 7, 10, 15, 20, 24 and 30 days. Duplicate pH 9 test solution vials were sampled at 0, 1. 7, 10, 17, 20, 24 and 30 days. With the exception of 0 day pH 5, both test solution vials were used for liquid scintillation counting (material balance), high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and pH measurement. Sterility measurements were conducted on only one of the test solution vials for each sampling interval. The test solutions maintained sterility and the pH was constant throughout the study.

The parent concentrations were essentially constant under pH 5, 7 and 9 conditions for the entire 30 day study period. No major degradate (10% of applied radioactivity) was formed under pH 5, 7 and 9 conditions. The material balance was 101.1 ± 1.9% under pH 5, 100.4 ± 1.4% under pH 7, and 104.2 ± 2.2% under pH 9 conditions. The calculated half-lives of the test substance using pseudo-first order reaction kinetics were 1406 days, 3136 days and 4101 days for pH 5, 7 and 9 conditions, respectively. The calculated rate constants of the test substance were 4.97E-04/day, 2.21E-04/day and -1.69E-04/day for pH 5, 7 and 9 conditions, respectively. The half-lives and the rate constants were calculated using HPLC data. The test substance has been shown to be hydrolytically stable under acidic, neutral, and basic conditions. The DT50 values were calculated to be 1406, 3136 and 4101 days in pH 5, pH 7 and pH 9 conditions, respectively. Based on these data, hydrolysis would not be expected to be the major degradative pathway for the test substance.