cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Aug 1999 to 28 Oct 1999

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 1981

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling: Duplicate samples were taken 0, 1, 2, 3, 4 and 5 days after treatment for pH 4, 5, 7 and 9. After sampling the pH was determined and thereafter for acidic and alkaline samples the pH adjusted to about 7 prior to storage or analysis.

- Preparation of Samples for Analyses: At appropriate time intervals test vessels were removed from the rotary water bath shaker, neutralised (pH 4, 5 and pH 9) and immediately handled (protecting from full light). If necessary, the samples were cooled down to ambient temperature. The pH value of each sample was measured using a calibrated electrode and the recovery of radioactivity determined by LSC. Thereafter, acidic and alkaline samples were neutralised, the radioactivity determined and aliquots submitted to HPLC or/and 2D-TLC.

- Storage: After neutralisation, all samples were stored at -20°C except for the time needed for analysis. The first HPLC analysis was done directly after sampling.

Buffers:

Suitable aqueous solutions/buffers were used. The buffers' ionic strength was ≤ 0.01 M to keep pH constant and buffer effects negligible. The dilution of the buffers was made with double distilled water.

The following buffer pH's and types were used:
pH 4 (0.01 M citrate)
pH 5 (0.01 M acetate)
pH 7 (0.01 M phosphate)
pH 9 (0.01 M borate)
The buffers/solutions were sterilised by sterile filtration; glassware was sterilised by autoclaving (121 °C, 40 min).

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks: Tightly sealed brown test tubes of 10 ml volume
- Sample volume: 5 mL
- Sample concentrations: 5 mg/L
- Sterility: To ensure sterile experimental conditions, all equipment was autoclaved at 120 ± 1 °C prior to use for 40 minutes, i.e. the hydrolysis vessels, gaskets and the laboratory glassware. All preparations of the test solutions were performed in a laminar flow sterile hood. The experimental buffers and solutions were sterilised by filtration prior to use.
- Type: Screw-capped sealed with a cover and a Teflon gasket to avoid volatilisation of the compound.
- Lighting: Protected from light during incubation and not handled in full light during sample analysis.
- Shaking condition: The flasks were placed in rotary water bath shakers and maintained at about 100 rpm shaking frequency.
- Temperature: 50 ± 0.5°C
- Temperature record: The rotary water bath shaker was thermostatically adjusted to maintain the temperature control of the test system flasks. For temperature recording, one representative brown test tube containing an aqueous solution corresponding to the test parameters was monitored for temperature variation by the incorporation of an indwelling thermocouple interface with a datalogger. Readings were recorded at 90 minutes intervals with an accuracy of 0.1 °C.

TEST MEDIUM
- Preparation of Stock Solution: For the preparation of the stock solution, 14C-triazole labelled test substance was dissolved in 10 mL acetonitrile. The radioactivity content was determined by LSC. The final concentration of the test substance in the stock solution was 2.84 mg a.i./10 mL. The test at pH 5 and 7 was repeated. For the repetition, a second stock solution was prepared containing 2.07 mg a.i./10 mL.
- Preparation of Application Solution: From the stock solution a volume of 8 mL was placed into 10 mL volumetric flask and the volume made up with acetonitrile to 10 mL. The total amount of radioactivity was determined to be 2.45 mg. The application solution for the repetition at pH 5 and 7 contained 1.08 mg a.i./10 mL.
- Treatment: For treatment an aliquot of the stock solution 2.045 mL at pH 4 and pH 9 or 2.4615 mL at pH 5 and 7 was placed into a sterile volumetric flask and the acetonitrile was removed under stream of nitrogen (sterile filtered). The residue was taken up in 100 mL of the corresponding sterile buffer solution. The final amount of radioactivity in the buffer solutions was detemined by LSC to be ca. 0.2 mg, corresponded to a test concentration of about 5 mg/L. From the corresponding treated buffer solutions aliquots of 5 mL each were placed into the test tubes. The treatment was performed under sterile conditions in a sterile hood.

Duration:

5 d

Temp.:

50 °C

Initial conc. measured:

5 mg/L

Remarks:

Applicable to all tested pHs (4, 5, 7 and 9)

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Preliminary study:

No degradation was observed in any of the buffers during the incubation period. (See Details on results)

Transformation products:

no

% Recovery:

> 100

pH:

4

Temp.:

50 °C

Duration:

5 d

% Recovery:

94.6

pH:

5

Temp.:

50 °C

Duration:

5 d

% Recovery:

94

pH:

7

Temp.:

50 °C

Duration:

5 d

% Recovery:

> 100

pH:

9

Temp.:

50 °C

Duration:

5 d

Key result

Remarks on result:

hydrolytically stable based on preliminary test

Details on results:

An overview of the results is provided in Table 1 – Table 4 in ‘Any other information on results incl. tables’

- Radiochemical Purity and Stability of the Test Substance: The radiochemical purity 14C-labelled test substance was determined to be 100 % by HPLC analysis. The stability of 14C-labelled test substance in the vehicle was proven by HPLC analysis after treatment. The test substance accounted for 100 % of radioactivity in the 0 day sample at pH 7 thus proving the stability of the test compound in the vehicle during treatment.

- Microbial Plate Counts: The samples prepared as positive control to prove the validity of the test showed, as expected, the presence of microorganisms. For the selected hydrolysis samples no colonies of microorganisms were found at the end of incubation (5 days) thus proving the sterility of the samples during the incubation period.

- pH of Test Solutions: The following pH's were observed: 4.2 ± 0.08, 5.2 ± 0.06, 7.2 ± 0.08, 9.0 ± 0.11

- Recovery of Radioactivity: The recovery was between 92.2 % and 103.1 % of total applied radioactivity at all four pH's (average of two replicates).

- Characterisation of Radioactivity in the Aqueous Buffer Solutions: No degradation of the parent compound was observed within the 5 days at 50 °C at pH 4, 5, 7 and 9 test substance accounted for 100 % of the radioactivity in the sample directly taken after treatment and at termination. The ratio of the two isomers of the test substance remained unchanged. The identity of the parent compound was confirmed by co-chromatography with reference standards (HPLC and 2D-TLC). The results of the HPLC analyses were confirmed by 2D-TLC

- Further test: The results of the pre-test showed that the test substance is hydrolytically stable, thus no further testing was performed.

Table 1. Degradation of the test substance in Buffer Solution pH 4 at 50 °C (Pre-test)

Incubation Time (Days)	Series	Test substance	Recovery
		% of region of interest (ratio of isomers A/B)	% of applied radioactivity
0	A	100.0 (49.9/50.1)	97.8
0	B	100.0 (48.9/51.1)	95.6
0	Mean	100.0	96.7
1	A	100.0	96.3
1	B	100.0	98.3
1	Mean	100.0	97.3
2	A	100.0	92.5
2	B	100.0	100.5
2	Mean	100.0	96.5
3	A	100.0	102.7
3	B	100.0	103.4
3	Mean	100.0	103.1
4	A	100.0	100.9
4	B	100.0	101.2
4	Mean	100.0	101.1
5	A	100.0 (49.1/50.9)	101.2
5	B	100.0 (48.7/51.3)	102.8
5	Mean	100.0	102.0

 

Table 2.Degradation of the test substance in Buffer Solution pH 5 at 50 °C (Pre-test)

Incubation Time		Test substance	Recovery
(Days)	Series	% of region of interest (ratio of isomers A/B)	% of applied radioactivity
0	A	100.0 (48.9/51.1)	95.7
0	B	100.0 (49.1/50.9)	98.8
0	Mean	100	97.3
1	A	100.0	97.8
1	B	100.0	98.1
1	Mean	100.0	98.0
2	A	100.0	93.2
2	B	100.0	93.6
2	Mean	100.0	93.4
3	A	100.0	94.6
3	B	100.0	94.0
3	Mean	100.0	94.3
4	A	100.0	92.2
4	B	100.0	92.2
4	Mean	100.0	92.2
5	A	100.0 (48.9 /51.1 )	95.0
5	B	100.0 (49.0/51.0 )	94.1
5	Mean	1 00.0	94.6

Table 3. Degradation of the test substance in Buffer Solution pH 7 at 50 °C (Pre-test)

Incubation Time		Test substance	Recovery
(Days)	Series	% of region of interest (ratio of isomers A/B)	% of applied radioactivity
0	A	100.0 (49.4/50.6)	97.3
0	B	100.0 (48.9/51.1)	97.8
0	Mean	100.0	97.6
1	A	100.0	97.1
1	B	100.0	97.2
1	Mean	100.0	97.2
2	A	100.0	93.3
2	B	100.0	93.7
2	Mean	100.0	93.5
3	A	100.0	93.4
3	B	100.0	93.8
3	Mean	100.0	93.6
4	A	100.0	93.3
4	B	100.0	94.4
4	Mean	100.0	93.9
5	A	100.0 (49.2/50.8)	93.8
5	B	100.0 (49.2/50.8)	94.1
5	Mean	100.0	94.0

 

Table 4. Degradation of the test substance in Buffer Solution pH 9 at 50 °C (Pre-test)

Incubation Time		Test substance	Recovery
(Days)	Series	% of region of interest (ratio of isomers A/B)	% of applied radioactivity
0	A	100.0 (49.1/50.9)	103.3
0	B	100.0 (48.9/51.1)	99.5
0	Mean	100.0	101.4
1	A	100.0	99.0
1	B	100.0	101.0
1	Mean	100.0	100.0
2	A	100.0	102.1
2	B	100.0	103.2
2	Mean	100.0	102.7
3	A	100.0	103.3
3	B	100.0	92.9
3	Mean	100.0	98.1
4	A	100.0	100.9
4	B	100.0	99.7
4	Mean	100.0	100.3
5	A	100.0 (49.4/50.6)	101.6
5	B	100.0 (49.3/50.7)	102.6
5	Mean	100.0	102.1

 

Validity criteria fulfilled:

yes

Conclusions:

No degradation was observed in any of the buffers during the incubation period. The substance was found hydrolytically stable under acidic, neutral and basic conditions.

Executive summary:

The hydrolysis of the substance was studied according to the OECD TG 111 and in compliance with GLP criteria. 14C-Triazole 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. 5 mg/L. The treated buffer test systems were incubated at 50 ± 0.5°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 every day after treatment for pH 4, 5 and 9 buffers. No significant hydrolysis of substance occurred during the entire incubation period for any of the buffer solutions. Thus, the test substance was found hydrolytically stable under all pre-test conditions and no further test was performed.

Description of key information

Hydrolytically stable under acidic, neutral, and basic conditions, OECD TG 111, Glänzel 1999.

Additional information

There are 2 studies available for this endpoint which followed standard guidelines. The study with four relevant pH values was selected as the key study. The other study (with three relevant pH value) is used as supporting information. In the key study (Glänzel 1999, Reliability 1), the 14C-Triazole 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. 5 mg/L. The treated buffer test systems were incubated at 50 ± 0.5°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 every day after treatment for pH 4, 5 and 9 buffers. No significant hydrolysis of substance occurred during the entire incubation period for any of the buffer solutions. Thus, the test substance was found hydrolytically stable under acidic, neutral, and basic conditions and no further test was performed. In the supporting study (Adam 2005, Reliability 1), the non-radiolabelled test item was applied to the sterile aqueous buffer solutions at pH 4, 7 and 9. The treated buffer test systems were incubated at 50 °C for up to 7 days. The results show that the test substance is hydrolytically stable under environmentally relevant acidic, neutral and alkaline conditions.