4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

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:

May 12, 1981

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

Two different [14C]-labeled test substances were used for the experiments: [phenyl-UL-14C]-trade name and [cyclohexyl-1-14C]-trade name

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: The sampling intervals were 0, 1,3, 7, 14, 21, 28, and 35 days, respectively.
- Sampling method: First, the vessels were opened, each 1 mL acetonitrile was added to the solutions intended for analyses, the vessels were closed again and manually shaken for about one minute. This procedure was found to be necessary in order to get better recoveries of RA.
- Sampling methods for the volatile compounds, if any: Volatilization of radioactivity from the solutions was not to be expected.
- Sampling intervals/times for pH measurements: The pH of test solutions incubated was measured after 1,14 and 35 days of incubation.
- Sampling intervals/times for sterility check: A subsample of each test solution was subjected to sterility check at day 1 (incubated at 25 °C, only) and after 14 and 35 days of incubation (both temperatures). About 15 µL were applied onto the mixed culture medium and incubated at room temperature in the dark for a minimum of 14 days.
- Sample storage conditions before analysis: Stored for not more than four days

Buffers:

- pH: 4 (citrate buffer), 7 (TRIS buffer), 9 (borate buffer)
- Type and final molarity of buffer: 0.01 M (all buffers)
- Composition of buffer: Chemicals of reagent grade quality from MERCK (or other manufacturer) were used to prepare the buffer solutions. Solvent used was highly purified water, processed through a Milli-Q unit (MILLIPORE) with bacterial filter. The resulting buffer solutions were diluted to the desired molarity of 0.01 with purified water and sterilized.

0.05 M Citrate buffer:
To 9 mL of a 0.1 N sodium hydroxide solution (4.0 g NaOH/L) 50 mL of a 0.1 M potassium dihydrogen citrate solution (23.02 g C6H7KO7/L) were added and diluted with water to a volume of 100 mL. The pH of this solution is measured with a glass electrode and then adjusted to pH 4.0 at the respective test temperature (25 ± 2 °C) using 0.1 N sodium hydroxide solution (4.0 g NaOH/l).

0.05 M TRIS buffer:
To 50 mL of a 0.1 M tris(hydroxymethyl)aminomethan solution (12.1 g C4H11NO3/L) 45 mL of an 0.1 N chloride acid solution (3.65 g HCI/L) were added. The pH of this solution is measured with a glass electrode and then adjusted to pH 7.0 at the respective test temperature (25 ± 2 °C) using 0.1 N sodium hydroxide solution or 0.1 N chloride acid solution.

0.02 M Boreate buffer:
0.04 moles boric acid (2.47 g H3BO3) were dissolved in 1 L of a 0.04 M potassium chloride solution (2.98 g KCI/L). To 125 mL of this solution 53 mL of a 0.04 M sodium hydroxide solution (1.6 g NaOH/L) were added and diluted with water to a volume of 250 mL. The pH of this solution is measured with a glass electrode and then adjusted to pH 9.0 at the respective test temperature (25 ± 2 °C) using 0.04 M sodium hydroxide solution or boric acid.

Details on test conditions:

TEST SYSTEM
- Sterilisation method: steam pressure sterilization
- Lighting: incubation in the dark

OTHER TEST CONDITIONS
- Adjustment of pH: yes

Duration:

35 d

pH:

4

Temp.:

25 °C

Initial conc. measured:

0.6 mg/L

Remarks:

Radioactivity [kBq/5 mL]: 11.3

Duration:

35 d

pH:

4

Temp.:

40 °C

Initial conc. measured:

0.6 mg/L

Remarks:

Label #1 Radioactivity [kBq/5 mL]: 11.3

Duration:

35 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.58 mg/L

Remarks:

Label #1 Radioactivity [kBq/5 mL]: 10.9

Duration:

35 d

pH:

7

Temp.:

40 °C

Initial conc. measured:

0.55 mg/L

Remarks:

Label #1 Radioactivity [kBq/5 mL]: 10.7

Duration:

35 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.6 mg/L

Remarks:

Label #1 Radioactivity [kBq/5 mL]: 11.3

Duration:

35 d

pH:

9

Temp.:

40 °C

Initial conc. measured:

0.6 mg/L

Remarks:

Label #1 Radioactivity [kBq/5 mL]: 11.3

Duration:

35 d

pH:

4

Temp.:

25 °C

Initial conc. measured:

0.6 mg/L

Remarks:

Label #2 Radioactivity [kBq/5 mL]: 11.7

Duration:

35 d

pH:

4

Temp.:

40 °C

Initial conc. measured:

0.6 mg/L

Remarks:

Label #2 Radioactivity [kBq/5 mL]: 11.7

Duration:

35 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.59 mg/L

Remarks:

Label #2 Radioactivity [kBq/5 mL]: 11.5

Duration:

35 d

pH:

7

Temp.:

40 °C

Initial conc. measured:

0.58 mg/L

Remarks:

Label #2 Radioactivity [kBq/5 mL]: 11.3

Duration:

35 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.58 mg/L

Remarks:

Label #2 Radioactivity [kBq/5 mL]: 11.7

Duration:

35 d

pH:

9

Temp.:

40 °C

Initial conc. measured:

0.58 mg/L

Remarks:

Label #2 Radioactivity [kBq/5 mL]: 11.7

Number of replicates:

18 vessels per label and temperature were used, i.e. 6 replicates per pH

Positive controls:

no

Negative controls:

no

Test performance:

- Sterility tests during the experiment demonstrated that sterile conditions could be maintained throughout the test period of the main hydrolysis experiment. Not any contamination was observed in the test solutions checked
- In the water bath of both the incubators the temperature was maintained constant throughout the study. The daily temperatures measured by means of a calibrated thermometer were on average 25.1 °C (RSD: 0.05; MAX: 25.2 °C; MIN: 25.0 °C) and 40.2 °C (RSD: 0.10; MAX: 40.4 °C; MIN: 40.1 °C), respectively.
- It is shown that the pH levels were kept at the set up values. A minor but not relevant change from 9.0 to 8.7 was determined in one vessel of the 40 °C test series of label #2 at day 35, only.

Transformation products:

yes

No.:

#1

No.:

#2

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

Quantification and characterization of the test item and its hydrolysis products:
Quantitative analyses of test solutions were done by thin-layer chromatography. For final evaluation solvent system B was used in case of radiolabel #1 and solvent system D in case of radiolabel #2. In general, similar conclusions resulted using the other solvent system each, but the hydrolysis products were not well to evaluate, then. Under the hydrolytic conditions of pH 4, 7 and 9 at 40 °C as well as at 25 °C both radiolabels showed degradation of the test item to each one main hydrolysis product. Further hydrolysis products (i.e. start zone RA, other peaks or remainder on TLC) were found to be not relevant (usually less than 2.5% of applied RA each). The unchanged test substance as well as the two main hydrolysis products were characterized by co-chromatography with the authentic reference substances, each. Furthermore, samples A-16 and B-17 (label #1) as well as A-17 and N-17 (label #2) were concentrated via Speed Vac®, investigated via HPLC and then analyzed via LC/MS. The concentrate of sample A-16 (label #1) was LC/MS analyzed under code TK 6. According to pos. ESI-Mode (yielded molecular ion at m/z 349) and to R(t) value (Ramona detector) the active ingredient amounted to about 52% of radioactivity in the preparative. Neither the CPT ((substance codes BNF 5572B or YRC 1689) nor the main further product in TK 6 (about 42% of radioactivity in the preparative) showed a yield of ions in the pos. ESI mode. But in the neg. ESI-Mode a quasi-molecular ion [M-H] at m/z 195 occurred. Furthermore, the R(t) values indicated that the main hydrolysis product of the [phenyl-UL-14C]-[trade name] was identical with BNF 5572B, corresponding to the compound 2-chlorophenyltetrazolinone (CPT). The concentrate of sample B-17 (label #1) was LC/MS analyzed under code TK 7. About 93% of radioactivity in that preparative were identified as CPT.
In the concentrates of samples A-17 and N-17 (label #2) which were LC/MS analyzed under code TK 8 and TK 9, the parent compound could be identified, only. Obviously, the predominant portions of the main hydrolysis product were lost during the preparation of the concentrates. Therefore, the main hydrolysis product observed when using label #2 was further characterized via sample code TK 10 got from the supplementary experiment. Sample TK 10 was a raw hydrolysis solution without concentrating in the vacuum. According to LC/MS and LC-MS/MS (pos. ESI-Mode: [M + H] at m/z 128) the substance cyclohexylethyl-amine (CEA, ref. code BNF 5578B) was identified, which amounted to about 51% of radioactivity in the preparative. Furthermore, the R(f) values on TLC and the R(t) values in HPLC confirmed that the main hydrolysis product of the [cyclohexyl-1-14C]-[trade name] was identical with the radiolabeled substance ECW 10409.
The test item is regarded as comparatively resistant to hydrolysis under acidic and neutral conditions at ambient temperatures. Under alkaline conditions the active ingredient is much faster degradable. From this study it is concluded that hydrolysis has some relevance for the degradation of the test item in the environment, especially in alkaline waters.

% Recovery:

99.2

pH:

4

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

100

pH:

7

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

97

pH:

9

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

98.5

pH:

4

Temp.:

40 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

98.8

pH:

7

Temp.:

40 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

97.7

pH:

9

Temp.:

40 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

98.6

pH:

4

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

98.5

pH:

7

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

97.8

pH:

9

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

99.4

pH:

4

Temp.:

40 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

100

pH:

7

Temp.:

40 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

% Recovery:

89

pH:

9

Temp.:

40 °C

Duration:

35 d

Remarks on result:

other: Mean recovery

Remarks:

For details see Table 2 below under "Any other information on results incl. tables"

Key result

pH:

4

Temp.:

25 °C

DT50:

319 d

Type:

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

Remarks on result:

other: far extrapolated

pH:

4

Temp.:

40 °C

DT50:

35 d

Type:

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

Key result

pH:

7

Temp.:

25 °C

DT50:

501 d

Type:

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

Remarks on result:

other: far extrapolated

pH:

7

Temp.:

40 °C

DT50:

36 d

Type:

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

Key result

pH:

9

Temp.:

25 °C

DT50:

69 d

Type:

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

Remarks on result:

other: extrapolated

pH:

9

Temp.:

40 °C

DT50:

9 d

Type:

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

Details on results:

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

MAJOR TRANSFORMATION PRODUCTS
At pH5 and pH7: Formation of each one relevant hydrolysis product, 2-chlorophenyltetrazolinone (CPT) in the case of [phenyl-UL-14C]-labelled test item and cyclohexylethylamine (CEA) in the case of [cyclohexyl-1-14C]-labelled test item, was observed in the tested solutions. After 35 days the observed hydrolysis products in the buffers pH 4 and pH 7 incubated at 25 °C were below 10%.
At pH9: Formation of each one relevant hydrolysis product, 2-chlorophenyltetrazolinone (CPT) in the case of [phenyl-UL-14C]-labelled test item and cyclohexylethylamine (CEA) in the case of [cyclohexyl-1-14C]-labelled test item, was observed in the tested solutions. After 35 days the observed hydrolysis products in buffer pH 9 were >20%, each.

VOLATILIZATION (at end of study)
The product CEA indicated a tendency to volatilize off the buffer.

PATHWAYS OF HYDROLYSIS
- Description of pathways: see attached Fig. 1
- Figures of chemical structures attached: Yes

Table 1: Time zero radioactivity and concentration of test substance in the buffers

Test solution ID	Radioactivity [kBq/5 mL]	Test item [mg/L]*
#1 A-00 for 25 and 40 °C	11.3	0.60
#1 N-00 for 25 °C	10.9	0.58
#1 N-00 for 40 °C	10.7	0.55
#1 B-00 for 25 and 40 °C	11.3	0.60
#2 A-00 for 25 and 40 °C	11.7	0.60
#2 N-00 for 25 °C	11.5	0.59
#2 N-00 for 40 °C	11.3	0.58
#2 B-00 for 25 and 40 °C	11.7	0.58

*: Label #1 evaluated acc. to solvent system B; Label #2 evaluated acc. to solvent system D

Table 2: Recovery of radioactivity in the different series of test vessels (5 mL buffer). [Based on the results of LSC a RA balance was established for each of the respective test vessels. The applied RA was defined as the amount of RA measured in the vessels taken just at beginning of the incubation period (day zero samples)].

Test solution ID	Recovery MEAN	Recovery MIN	Recovery MAX	Trend during Incubation time
#1 pH 4 incubated at 25 °C	99.2	98.0	99.7	+ -
#1 pH 7 incubated at 25 °C	100.0	97.9	104.3	+ -
#1 pH 9 incubated at 25 °C	97.0	92.0	99.1	+ -
#1 pH 4 incubated at 40 °C	98.5	95.8	101.3	+ -
#1 pH 7 incubated at 40°C	98.8	59. 8*	107.0	+-
#1 pH 9 incubated at 40°C	97.7	96.0	99.1	+ -
#2 pH 4 incubated at 25 °C	98.6	96.5	100.8	+ -
#2 pH 7 incubated at 25 °C	98.5	93.9	100.3	+ -
#2 pH 9 incubated at 25 °C	97.8	95.9	99.3	(+) -
#2 pH 4 incubated at 40 °C	99.4	98.7	100.2	+ -
#2 pH 7 incubated at 40°C	100.4	95.3	102.8	+ -
#2 pH 9 incubated at 40 °C	89.0	81.2	99.2	-

*: Value 59.8 regarded as outlier

Based on the results of LSC a RA balance was established for each of the respective test vessels. The applied RA was defined as the amount of RA measured in the vessels taken just at beginning of the incubation period. Generally, complete material balances were found (except one outlier, the vessel N-01, 40 °C). But there was a clear tendency (especially observed at 40 °C in buffer pH 9) of a decreasing recovery of RA in the longer incubated test solutions in the case of the cyclohexyl-1-14C-label #2. Therefore, the test solution of vessel B-17 was investigated in order to clarify the observed loss. The gathered data (LSC and HPLC analyses of solution before and after purging it by nitrogen) indicated that the incomplete RA balances were caused by volatilization of portions of the formed main hydrolysis product CEA (corresponding to sample ID ECW 10409) out of the test solution.

Table 3: Radioactivity balance for the vessels with [cyclohexyl-1-14C]-label #2 incubated at 40 °C

Sample code	Incubation time [days]	Radioactivity in solution [Bq/5 mL]*	Recovery [%]
A-00	0.0	11,668	100.00
A-11	1.0	11,550	98.99
A-12	3.0	11,560	99.07
A-13	7.0	11,631	99.68
A-14	14	11,633	99.70
A-15	21	11,586	99.30
A-16	28	11,694	100.22
A-17	35	11,510	98.65
N-00	0.0	11,284	100.00
N-11	1.0	10,853	95.29
N-12	3.0	11,294	100.09
N-13	7.0	11,374	100.80
N-14	14	11,448	101.45
N-15	21	11,450	101.47
N-16	28	11,602	102.82
N-17	35	11,366	100.73
B-00	0.0	11,713	100.00
B-11	1.0	11,614	99.15
B-12	3.0	10,566	90.21
B-13	7.0	10,615	90.63
B-14	14	10,926	93.28
B-15	21	10,094	86.18
B-16	28	9,650	82.39
B-17	35	9,514	81.23

* Measured by each three 100 µL aliquots taken of a final volume of 6 mL (5 mL incubated sample plus 1 mL acetonitrile)

Table 4: Kinetic results (1st order) of the test item hydrolysis [Only in case of 40 °C the testing period of 35 days was sufficient to degrade at least half of the parent compound. The given longer half-lives are extrapolated values]

Test solution (buffer)	Temperature	Half-life [days]
		Label #1	Label #2	Mean
pH 4 (0.01 M citrate buffer)	25 °C	289.2	357.2	319
	40 °C	31.8	38.6	35
pH 7 (0.01 M TRIS buffer)	25 °C	663.9	391.4	501
	40 °C	32.5	38.6	36
pH 9 (0.01 M borate buffer)	25 °C	62.2	77.7	69
	40 °C	8.8	9.7	9

The study demonstrated the hydrolytic stability of the test item at 25 °C in pure buffers of pH 4 and 7. Since the experimental data had to be extrapolated far beyond the test period, it is not at all surprising that the single values have a greater discrepancy. However, in buffer pH 9 a significant degradation (about 33%) was measured during the testing period of 35 days and the gathered data of different test series were comparable. As it was slightly found at 25 °C already, the tests at 40 °C resulted in a constant decrease of the concentration of the test item in the test solutions. 

Validity criteria fulfilled:

not applicable

Description of key information

Hydrolysis may contribute to the transformation of the substance under alkaline environmental conditions.

Additional information

One study is available testing the potential for hydrolysis of the test substance (1997). The test follows the principles of the OECD guideline 111 and was conducted under GLP standards. The hydrolysis of the test item was studied in three 0.01 M buffer solutions adjusted to pH 4 (citrate), pH 7 (TRIS) and pH 9 (borate). Two different 14C-labeled test substances each diluted to about 0.6 mg/L buffer were used for the experiments. The test solutions were incubated in the dark for a maximum period of 35 days under sterile conditions at 25 °C or at 40 °C. The sampling intervals were 0, 1, 3, 7, 14, 21, 28, and 35 days, respectively. Formation of two relevant hydrolysis products was observed in the tested solutions. After 35 days the observed hydrolysis products, Cyclohexylethylamine (CEA) and 2-Chlorophenyltetrazolinone (CPT), in the buffers pH 4 and pH 7 incubated at 25 °C were below 10%, but in buffer pH 9 they were >20%, each. One hydrolysis product indicated a tendency to volatilize off the buffer. The respective calculated 1st order half-lives of the test item in the tested buffers were:

Test solution (buffer)	Temperature	Calculated half-life [days] (mean of both labels)
pH 4	25 °C 40 °C	319 (far extrapolated) 35
pH 7	25 °C 40 °C	501 (far extrapolated) 36
pH 9	25 °C 40 °C	69 (extrapolated) 9

The test item is regarded as comparatively resistant to hydrolysis under acidic and neutral conditions at ambient temperatures. Under alkaline conditions the active ingredient is much faster degradable. From this study it is concluded that hydrolysis has some relevance for the degradation of the test item in the environment, especially in alkaline waters.