1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Feb 2004 to 5 May 2004

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:

Draft, August 2001

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147, Agricultural Production Bureau

Version / remarks:

revised 26th June 2001

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-triazolyl labelled

Analytical monitoring:

yes

Details on sampling:

- The definitive sampling regimes are summarised in Table 2 in “Any other information on materials and methods incl.tables”. The pH 7 samples were analysed separately because the first pH 7 buffer was not close enough to the required value and a new buffer was prepared. The additional sampling intervals refer to extra analyses conducted on the pH 4, 5 and 9 buffers because of problems with the temperature printer. These analyses were included to confirm that the original acquired data was correct.

Buffers:

The buffer solutions were prepared using ultra-pure water as seen in Table 1, in “Any other information on materials and methods incl. tables”. The concentration of each buffer was < 0.1M to maintain constant pH whilst minimising buffer effects. Each buffer was sterilised by autoclaving at 120°C. Prior to application, 25mL aliquots of the appropriate buffer solution were transferred into the required number of Pierce amber hypovials (30 mL). The amber hypovials were used to minimise the effect of light.

Details on test conditions:

PREPARATION OF TREATMENT SOLUTION
The radiochemical was received in a solution of acetonitrile. For use, the radiochemical was quantitatively transferred to a volumetric flask (2 mL) and diluted with acetonitrile to give a final volume of 2 mL. The radioactive content of the stock solution was quantified by LSC. Sterilisation of the treatment stock solution was considered unnecessary as the acetonitrile would inhibit microbial growth.

APPLICATION OF RADIOCHEMICAL TO BUFFER TEST SYSTEMS
The 14C-substance treatment solution (250 µL) was added to each sterilized Pierce amber hypovial (30 mL) containing the buffer solutions, using a Gilson Microman positive displacement pipette, to give a final concentration of ca. 10 µg/mL. After treatment, the vessels were gently swirled to ensure complete mixing. 1.5 mL aliquots were removed and transferred to the appropriate sterilized autosampler vial, which were then sealed before incubation in the water bath. The vial caps were tightly closed and the headspace was minimised by nearly filling the HPLC vials to limit evaporation losses. Amber vials were used to prevent light effects. These procedures were conducted inside a Class II laminar flow Microbiology cabinet to maintain sterility. Homogeneity checks were carried out pre- and post-application by removing 100 or 50 µL aliquots for LSC quantification during each application procedure.

INCUBATION, MONITORING OF TEMPERATURE AND pH MEASUREMENT
The treated test systems were incubated in a Grant water bath at 50 ± 1°C in the absence of light. The temperature of the test systems was monitored, by immersing an AK29M or AK29 thermocouple into the water, the temperature being recorded using a KM1242 temperature printer at a half hourly interval. The pH of the buffers was measured pre-application and at each sampling time using a Knick pH meter connected to a Hamilton electrode. The pH meter was checked using Hanna Instruments pH 4, 7 and 10 standard buffer solutions.

STERILITY
Sterile conditions are required for hydrolysis studies in order to prevent potential microbial degradation of the test substance. Aseptic techniques were used throughout the preparation of the samples within a Class II Microbiology cabinet to ensure sterility was maintained. Glassware, such as the hypovials, was sterilised by autoclaving at 120°C. Equipment used that could not be sterilised by autoclaving (e.g. pipette tips, autosampler vials) was soaked overnight in an ethanol;water (70:30 v/v) solution. Sterility checks were carried out by dispensing 0.5 mL aliquots of the designated treated buffer in duplicate onto pre-poured nutrient agar plates. “Clean controls” were also prepared by exposing the plates to the environment of the Class II cabinet without the addition of sample, in order to provide an indication of background levels of contamination. “Dirty controls” were produced by touching the surface of the agar plate with a bare finger to expose the agar to microbial contamination. This control showed that the prepared agar plates were capable of developing microbial colonies. The plates were incubated at 20 ± 2°C, and inspected visually for microbial growth after 7 days incubation.

ANALYSIS OF TREATED TEST VESSELS
- At each sampling time point, the HPLC vials were removed from the water bath and the radioactive content of the treated buffers was determined by LSC by transferring suitable aliquots of the aqueous solution to LSC vials for counting (The 0 day samples were not analysed by LSC since the homogeneity checks were used to determine the application rates and no further manipulations were conducted on the samples). The remainder of the buffer solution in the HPLC vial was analysed by HPLC with radio-detection, to determine the extent of degradation of the substance.

SAMPLE HANDLING AND STORAGE
Samples were analysed by LSC on the day they were generated and within 24 hours for HPLC analysis. The treatment solution was stored frozen when not in use. The remaining buffer solution that was not transferred to the HPLC vials was also stored frozen. The treated samples in HPLC vials were stored at <8°C after analysis.

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

10 mg/L

Number of replicates:

Duplicate

Positive controls:

no

Negative controls:

no

Preliminary study:

Not specified

Test performance:

Not reported

Transformation products:

not measured

% Recovery:

>= 98.2 - <= 98.6

pH:

4

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

% Recovery:

>= 99.7 - <= 100

pH:

5

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

% Recovery:

99

pH:

7

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

% Recovery:

>= 98.3 - <= 99.5

pH:

9

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

Key result

Temp.:

20 °C

DT50:

> 1 yr

Remarks on result:

hydrolytically stable based on preliminary test

Details on results:

RADIOCHEMICAL PURITY AND HOMOGENEITY CHECKS
- The mean radiochemical purities at the time of the applications, are summarised in Table 1. The mean radiochemical purity was >98.5% and there was no single radiochemical impurity >1%. This was considered adequate for the purposes of this study. Data from the LSC counts of the homogeneity check aliquots confirmed that the treatment solution remained homogeneous throughout the application.

APPLICATION RATE
- The application rate was calculated from the mean value of the pre- and postapplication homogeneity checks samples for each of the incubation conditions. The intended application rate was 10 µg mL-1 (10 ppm) and the actual application rates were between 10.0 and 10.1 µg mL-1. The application rate for each incubation condition is summarised in Table 2.

TEMPERATURE AND pH MEASUREMENTS
- The recorded temperature of the water bath was consistent throughout the study, ranging from 49.9 to 51.1°C (the average temperature = 50.5°C). There were four instances when the temperature exceeded the 50 ± 1°C range. However, the upper limit was only slightly exceeded (51.1°C) and the temperature was within the required range by the next reading. Problems with the printer resulted in the temperature not being recorded for certain periods, however additional incubations were added to support the data already produced. The measured pH values of each buffer for the pre-application solutions and at each sampling point are displayed in Table 3. The pH values for the buffers were considered close enough to their required values and were consistent throughout the course of the incubation period.

STERILITY
- No significant microbial growth was detected on any of the nutrient agar plates for the treated samples or the “clean controls”, confirming that sterility had been maintained throughout the entire incubation period for buffers treated with radiolabelled test material. Growth was apparent on the “dirty controls” however, showing that the nutrient agar plates were suitable for growth.

TOTAL RECOVERY OF APPLIED RADIOACTIVITY
- The total amount of radioactivity recovered (or mass balance) from each test vessel was determined from the activity found by LSC quantification at each sampling time point. The mass balance ranged from 98.2% to 101.9% with an overall recovery of 99.6% for all treated vessels. (These values are derived from the 2, 3 and 5 DAT samples at pH values 4, 5, 7 and 9). The data is summarized in Table 4. The total amount of radioactivity recovered for the additional sampling intervals (pH buffers 4, 5 and 9 only) is shown in Table 5.

RADIOACTIVE RESIDUES IN THE TREATED TEST VESSELS
- The aqueous buffers were directly analysed by HPLC. The results demonstrated that the applied 14C-substance was stable in all of the buffers (pH 4, 5, 7 and 9), and therefore no degradates were observed. The data for each treated test vessel is summarised in Table 6 and for the additional intervals in Table 5. There was no significant degradation in any of the additional sampling intervals. No adjustments were made for the purity of the application solution. Slight shifts in retention times were observed during the study due to problems with the timer of the radio-detector.

LC-MS-MS CONFIRMATION OF THE SUBSTANCE
- Selected samples were analysed by LC-MS-MS to confirm the presence of the substance. Comparison of the 0 DAT and 5 DAT samples for each buffer with the authenticated reference standard using electrospray in positive ion mode confirmed that the compound was the substance.

Table 1. Radiochemical Purities of ¹⁴C-Substance Treatment Solutions

Experiment	Mean purity (HPLC)
Pre-application (pH 4, 5 and 9)	98.6%
Pre-application (pH 7)	98.8%

 

Table 2. ¹⁴C-Substance Added to Test Vessels

pH Buffer	Total Amount of Radioactivity (Bq) Applied (25 mL)	Dose Rate (ppm)
4	516750.0	10.1
5	510162.5	10.0
7	516437.5	10.1
9	513337.5	10.0

The specific activity of ¹⁴C-substance is 2.05 MBq mg^-1

 

Table 3. Measurement of pH of the Buffer Solutions

Sampling Interval (Days)	Buffer Solutions
	pH 4	pH 5	pH 7	pH 9
Pre-Application	3.88	4.92	6.93	9.22
0	3.89	4.94	6.93	9.23
3	3.88	4.90	6.99	9.23
5	3.92	4.91	6.96	9.21

 

Table 4. Summary of Quantitative Results Obtained from Hydrolysis of the substance for Each Buffer

a) pH 4 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100^(1)	98.1
	100^(1)	99.0
3	99.5	98.5
	101.0	99.9
5	98.2	97.2
	98.6	97.3

 (b) pH 5 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100^(1)	98.4
	100^(1)	98.4
3	101.9	101.4
	101.8	101.8
5	99.7	99.0
	101.1	98.7

 (c) pH 7 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100^(1)	98.4
	100^(1)	98.7
3	98.5	97.9
	98.2	97.3
5	99.0	98.2
	99.0	98.3

 (d) pH 9 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100^(1)	98.8
	100^(1)	98.1
3	100.5	99.4
	99.4	98.2
5	98.3	97.3
	99.5	98.0

(1) -The 0 DAT samples were not analysed by LSC, the recovery was calculated from the mean value of the pre- and post-homogeneity checks.

 

 

Table 5. Additional Sampling Intervals: Summary of Quantitative Results Obtained from Hydrolysis of the substance for the Buffers at pH 4, 5 and 9.

(a) pH 4 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100	98.1
		98.8
5	105.8	104.7
	105.7	104.5

 (b) pH 5 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100	99.0
		98.6
5	113.6	112.6
	114.6	113.5

 (c) pH 9 Buffer

Time Interval (Days)	% Recovery	% Substance
0	100	98.7
		98.2
5	96.3	95.3
	100.3	99.4

Validity criteria fulfilled:

yes

Conclusions:

14C-Triazolyl labelled substance was applied to sterile aqueous buffers at pH 4, 5, 7 and 9 and the buffers were incubated in the dark at 50 ± 1°C for up to 5 days. No degradation was observed in any of the buffers and, therefore, substance was hydrolytically stable under acidic, neutral and basic conditions at environmentally relevant temperatures. Sterile conditions were maintained throughout the incubation periods.

Executive summary:

The hydrolysis of the substance was studied according to the OECD TG 111 and in compliance with GLP. ¹⁴C-Triazolyl labelled substance was applied to sterile aqueous buffer solutions at pH 4, 5, 7 and 9 contained in sealed amber vials to achieve a concentration of ca. 10 µg/mL. The treated buffer test systems were incubated at 50 ± 1°C for up to 5 days. The test systems were incubated in the absence of light and sterile conditions were maintained throughout the study. Samplings were carried out at 0, 3 and 5 days (for pH 4, 5 and 9 buffers) and at 0, 2 and 5 days (for pH 7 buffer) after treatment. No significant hydrolysis of substance occurred at 50 °C in the 5 day incubation period for any of the buffer solutions. Compounds exhibiting 10% hydrolysis at 50 °C over 5 days would have half-lives of 25 days, which is equivalent to a value of approximately 1 year at 20°C. Test substances with less than 10% hydrolysis over 5 days at 50°C are considered hydrolytically stable and no further testing is necessary at low temperatures. In conclusion, this study demonstrated that substance is hydrolytically stable at pH 4, 5, 7 and 9 at environmentally relevant temperatures.

Description of key information

Hydrolytically stable under acidic, neutral and basic conditions, environmentally relevant temperatures, OECD TG 111, Oliver & Edwards 2004 

Additional information

There is one hydrolysis study available, which followed OECD TG 111 and in compliance with GLP criteria. 14C-Triazolyl labelled substance was applied to sterile aqueous buffer solutions at pH 4, 5, 7 and 9 contained in sealed amber vials to achieve a concentration of ca. 10 µg/mL. The treated buffer test systems were incubated at 50 ± 1°C for up to 5 days. The test systems were incubated in the absence of light and sterile conditions were maintained throughout the study. The study demonstrated that the substance is hydrolytically stable at pH 4, 5, 7 and 9 at environmentally relevant temperatures.