Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jan 2006 to 29 Jun 2006

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:

11 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Version / remarks:

18 October 1982/ and Addendum 3, PB88-159892, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling (pre-test): performed at 50 °C, duplicate samples were taken at 0, 1, 2, 3, 4 and 5 days after treatment for pH 4, 5, 7 and 9.
- Confirmatory EPA test: performed at 25 °C, duplicate samples were taken at 0, 15 and 30 days after treatment at pH 5, 7 and 9.
- Sampling intervals/times for sterility check: Sterility was confirmed at Day 0 and at study termination.
- Sample storage conditions before analysis: All samples were extracted on the day of sampling.
- Storage stability : Since the samples were analysed by HPLC within one week, no storage stability validation was performed.

Buffers:

- pH 4 (0.01 M Citrate buffer): 0.0559 mole citric acid, 0.0439 mole hydrogen chloride and 0.1120 mole sodium hydroxide are in in 1 L Tritisol-buffer Merck. An aliquot of the Tritisol-buffer (25 mL) was transferred to volumetric flask (250 mL) and brought to volume with water.
- pH 5 (0.01 M acetate buffer): 0.825 mol acetic acid and 1.795 mole sodium acetate are in 1 L acetate buffer Fluka. An aliquot of the acetate-buffer (3.8 mL) was transferred to volumetric flask (1000 mL) and brought to volume with water then at pH 5 adjusted.
- pH 7 (0.01 M phosphate buffer): 0.069 mole phosphate buffer: 0.028 mole potassiumdihydrogenphosphate and 0.041 mole disodium hydrogenphosphate are in 1 L phosphate buffer. An aliquot of the phosphate-buffer (145 mL) was transferred to volumetric flask (1000 mL) and brought to volume with water.
- pH 9 (0.01 M borate buffer): 0.017 mole potassiumdihydrogenphosphate and 0.043 mole disodiumtetraborate are in 1 L borax-buffer. An aliquot of the borax-buffer (167 mL) was transferred to volumetric flask (1000 mL) and brought to volume with water.

Details on test conditions:

TEST SYSTEM
- Test vessel: Tightly sealed brown test tubes
- Sterilisation method: The buffers/solutions were sterilised by sterile filtration; glassware was sterilised by autoclaving (121°C, 60 min). All preparations of the test solutions were performed in a laminar flow sterile hood.
- Lighting: Darkness
- Measures taken to avoid photolytic effects: Samples were protected from light during incubation and not handled in full light during sample analysis

TEST MEDIUM
- Volume used/treatment: 0.05 mL
- Preparation of test solution: [Pyrazole-5-14C]-labelled test substance was dissolved in 5 mL acetonitrile. The radioactivity content was determined by LSC. The final concentration of the test item in the stock solution was 2.2 mg/ 5 mL. For the application solution, 0.70 mL of the previous stock solution was placed into a sterile 10 mL volumetric flask and was filled with acetonitrile. The radioactivity content was determined by LSC. The final concentration of the test item in the application solution was 0.325 mg/ 10 mL.
- Tretament: For treatment an aliquot of the application solution (0.05 mL) was placed into each sterile 5 mL volumetric flask and the acetonitrile was removed under a stream of nitrogen (sterile filtered). The residue was taken up in 5 mL of the corresponding sterile buffer solution. The final amount of radioactivity in the buffer solutions was determined by LSC to be ca. 0.0016 mg, corresponded to a test concentration of approximately 0.32 mg/L.

OTHER TEST CONDITIONS
- Adjustment of pH: For acidic and alkaline samples, the pH was adjusted to about 7 prior to storage or analysis.

Duration:

5 d

Temp.:

50 °C

Initial conc. measured:

0.32 mg/L

Remarks:

Pre-test; pH 4, 5, and 7

Duration:

30 d

Temp.:

25 °C

Initial conc. measured:

0.32 mg/L

Remarks:

Confirmatory EPA test; pH 5, 7 and 9

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Preliminary study:

An overview of the results is provided in Table 1 and Table 3 in ‘Any other information on results incl. tables’.
- Recovery of radioactivity: The recovery of the pre-test was between 90.9 % and 99.3 % of total applied radioactivity at all four pH values (average of two replicates).
- Characterisation of radioactivity in the aqueous buffer solutions: No degradation of the parent compound was observed at 50oC at pH 4, 5, 7 and 9 during the test period of 5 days. The test substance as sum of both isomers syn- and anti-isomers accounted for 97.7 % of the radioactivity in the sample directly taken after treatment and for 92.6 % at termination at pH 4. The corresponding values were 97.2 % and 92.3 % at pH 5, 93.2 % and 93.9 % at pH 7 and 98.6 % and 97.2 % at pH 9. The identity of the parent compound or the two isomers was confirmed by co-chromatography with the reference item (HPLC and 2D-TLC). The results of the HPLC analyses were confirmed by 2D-TLC.

Transformation products:

not specified

Remarks:

Unknown

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

One minor degradate was formed, not exceeding 1.5% of the applied radioactivity at any time point throughout the preliminary or confirmatory tests.

% Recovery:

94.3

St. dev.:

2.6

pH:

4

Temp.:

50 °C

Duration:

5 d

% Recovery:

94.5

St. dev.:

2.3

pH:

5

Temp.:

50 °C

Duration:

5 d

% Recovery:

93.6

St. dev.:

2.2

pH:

7

Temp.:

50 °C

Duration:

5 d

% Recovery:

96.1

St. dev.:

2.7

pH:

9

Temp.:

50 °C

Duration:

5 d

% Recovery:

93.8

St. dev.:

3.4

pH:

5

Temp.:

25 °C

Duration:

30 d

% Recovery:

94.4

St. dev.:

3.6

pH:

7

Temp.:

25 °C

Duration:

30 d

% Recovery:

95.3

St. dev.:

5

pH:

9

Temp.:

25 °C

Duration:

30 d

Key result

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

other: Arrhenius equation

Remarks on result:

other: < 10% of hydrolysis is observed at 50°C after 5 days

Details on results:

An overview of the results is provied in Table 1 - Table 3 in 'Any other information on results incl. tables'
- Radiochemical purity and stability of the test item: The radiochemical purity 14C-labelled test substance was determined to be 97.9 % (sum of syn and anti) 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 (sum of syn and anti) thus proving the stability of the test compound in the vehicle during treatment.
- Microbial plate counts: For the selected hydrolysis samples no colonies of micro- rganisms were found at the end of incubation (5 days or 30 days) thus proving the sterility of the samples during the incubation period.
- Final test: The recovery of the EPA confirmatory test ranged from 91.2% to 99.3% (average of two replicates). No degradation of the parent compound was observed after 30 days at pH 5, 7 and 9. The test substance as sum of both syn- and anti- isomers accounted for 97.2 % of the radioactivity in the sample directly taken after treatment and for 91.5 % at termination at pH 5. The corresponding values were 93.2 % and 92.8 % at pH 7, 99.3 % and 95.6 % at pH 9.

Table 1. Mass Balance and distribution of radioactivity at 50ºC (preliminary test) – individual replicates (values as % of applied)

pH	Fraction	Rep.	Incubation time (days)
			0	1	2	3	4	5
4	Syn isomer (Parent)	A	91.1	86.2	82.8	82.8	84.4	81.8
		B	86.1	90.7	89.1	82.7	87.1	88.7
		mean	88.6	88.5	86.0	82.8	85.8	85.3
	Anti isomer (Parent)	A	9.0	7.6	8.0	8.2	7.2	7.3
		B	9.2	6.6	8.0	8.2	8.2	7.4
		mean	9.1	7.1	8.0	8.2	7.7	7.4
	Parent Total	A	100.1	93.8	90.8	91.0	91.6	89.1
		B	95.3	97.3	97.1	90.9	95.3	96.1
		mean	97.7	95.6	94.0	91.0	93.5	92.6
	Unknown	A	*	*	*	*	0.9	1.8
		B	*	*	*	*	*	*
		mean	*	*	*	*	0.5	0.9
	Recovery	A	100.2	93.9	90.8	90.9	92.4	90.9
		B	95.3	97.4	97.0	90.9	95.3	96.1
	Mean ± SD		94.3 ± 2.6
5	Syn isomer (Parent)	A	87.3	87.0	87.2	85.1	87.6	84.0
		B	90.9	85.9	88.9	82.8	88.9	86.0
		mean	89.1	86.5	88.1	84.0	88.3	85.0
	Anti isomer (Parent)	A	7.7	7.0	7.2	8.6	7.3	8.1
		B	8.4	7.7	7.7	8.0	7.2	6.5
		mean	8.1	7.4	7.5	8.3	7.3	7.3
	Parent Total	A	95.0	94.0	94.4	93.7	94.9	92.1
		B	99.3	93.6	96.6	90.8	96.1	92.5
		mean	97.2	93.8	95.5	92.3	95.5	92.3
	Unknown	A	*	*	*	*	*	0.7
		B	*	*	*	*	*	*
		mean	*	*	*	*	*	0.4
	Recovery	A	95.0	94.0	94.5	93.7	94.9	92.8
		B	99.3	93.6	96.6	90.7	96.1	92.5
	Mean ± SD		94.5 ± 2.3
7	Syn isomer (Parent)	A	84.3	83.3	85.8	84.1	83.1	84.2
		B	84.6	89.7	88.7	84.5	84.8	87.5
		mean	84.5	86.5	87.3	84.3	84.0	85.9
	Anti isomer (Parent)	A	8.9	7.6	7.8	8.4	7.7	8.3
		B	8.6	8.1	7.9	8.5	7.3	7.7
		mean	8.8	7.9	7.9	8.5	7.5	8.0
	Parent Total	A	93.2	90.9	93.6	92.5	90.8	92.5
		B	93.2	97.8	96.6	93.0	92.1	95.2
		mean	93.2	94.4	95.1	92.8	91.5	93.9
	Unknown	A	*	*	*	1.9	*	*
		B	*	*	*	*	*	*
		mean	*	*	*	1.0	*	*
	Recovery	A	93.2	90.9	93.6	94.4	90.8	92.5
		B	93.1	97.8	96.7	93.0	92.1	95.2
	Mean ± SD		93.6 ± 2.2
9	Syn isomer (Parent)	A	91.2	88.6	85.3	86.8	89.8	89.1
		B	89.8	88.1	86.8	83.3	84.5	87.4
		mean	90.5	88.4	86.1	85.1	87.2	88.3
	Anti isomer (Parent)	A	8.4	8.0	8.2	7.8	8.0	9.2
		B	7.7	8.2	7.5	7.7	7.0	8.6
		mean	8.1	8.1	7.9	7.8	7.5	8.9
	Parent Total	A	99.6	96.6	93.5	94.6	97.8	98.3
		B	97.5	96.3	94.3	91.0	91.5	96.0
		mean	98.6	96.5	93.9	92.8	94.7	97.2
	Unknown	A	0.6	1.4	*	*	*	0.6
		B	0.8	1.5	*	0.5	*	*
		mean	0.7	1.5	*	0.3	*	0.3
	Recovery	A	100.7	97.9	93.5	94.6	97.8	98.9
		B	98.3	97.8	94.2	91.6	91.5	96.1
	Mean ± SD		96.1 ± 2.7

 

Table 2. Mass Balance and distribution of radioactivity at 25ºC (confirmatory test/ EPA test) – individual replicates (values as % of applied)

pH	Fraction	Rep.	Incubation time (days)
			0	15	30
5	Syn isomer (Parent)	A	87.3	83.2	83.7
		B	90.9	87.2	84.1
		mean	89.1	85.2	83.9
	Anti isomer (Parent)	A	7.7	7.1	6.9
		B	8.4	8.0	8.3
		mean	8.1	7.6	7.6
	Parent Total	A	95.0	90.3	90.6
		B	99.3	95.2	92.4
		mean	97.2	92.8	91.5
	Recovery	A	95.0	90.2	90.6
		B	99.3	95.2	92.3
	Mean ± SD		93.8 ± 3.4
7	Syn isomer (Parent)	A	84.3	86.7	84.3
		B	84.6	91.4	85.2
		mean	84.5	89.1	84.8
	Anti isomer (Parent)	A	8.9	6.7	8.0
		B	8.6	9.9	8.0
		mean	8.8	8.3	8.0
	Parent Total	A	93.2	93.4	92.3
		B	93.2	101.3	93.2
		mean	93.2	97.4	92.8
	Recovery	A	93.2	93.3	92.3
		B	93.1	101.2	93.2
	Mean ± SD		94.4 ± 3.6
9	Syn isomer (Parent)	A	91.4	82.8	91.2
		B	89.7	83.6	82.5
		mean	90.6	83.2	86.9
	Anti isomer (Parent)	A	8.8	8.1	9.7
		B	8.7	7.9	7.7
		mean	8.8	8.0	8.7
	Parent Total	A	100.2	90.9	100.9
		B	98.3	91.5	90.2
		mean	99.3	91.2	95.6
	Recovery	A	100.2	90.9	100.9
		B	98.3	91.5	90.2
	Mean ± SD		95.3 ± 5.0

Table3. Confirmation Of HPLC Results By 2D-TLC Analysis on test substance

	HPLC	2D-TLC
Samples	% of recovered radioactivity
5 days series A at pH 4	100.0	100.0
30 days series A at pH 9	100.0	100.0

Validity criteria fulfilled:

yes

Conclusions:

In a hydrolysis study performed in accordance with OECD TG 111 and EPA 161-1, Less than 10% hydrolysis of test substance was observed for all four pH values after 5 days at 50 °C (equates to a DT50 > 1 year at 25 °C). The test substance was confirmed to be hydrolytically stable at pH 5,7 and 9 at 25 ˚C for 30 days. Thus, it was concluded that the substance is hydrolytically stable under acidic, neutral and alkaline conditions.

Executive summary:

The hydrolysis of the test substance was studied according to OECD TG 111 and EPA 161 -1 guidelines and complied with GLP criteria. In the preliminary test, sterile aqueous buffer solutions (pH 4, pH 5, pH 7 and pH 9) were prepared containing 0.32 mg/L [14C-pyrazole]-labelled test substance. Each treated buffer was then incubated at 50°C in sealed vessels with no facility for trapping CO2 and organic volatiles for up to 5 days in the dark. Duplicate samples were taken daily for analysis during the incubation by LSC and HPLC. In a confirmatory test, the 14C-labelled test substance was incubated at pH 5, 7 and 9 at 25˚C for 30 days in the dark. Duplicate samples were taken on day 0, 15 and 30 during the test.

In the preliminary test at 50 °C, the mean recoveries of radioactivity ranged from 90.9% to 99.3% of the applied radioactivity (AR) for all pH values. In the confirmatory test at 25 °C, the corresponding range was 91.2% to 99.3% AR. The test substance was stable to hydrolysis at all four pH values. Less than 10% hydrolysis of test substance was observed for all four pH values after 5 days at 50 °C (equates to a DT50 > 1 year at 25 °C). The test substance was confirmed to be hydrolytically stable at pH 5, 7 and 9 at 25 ˚C for 30 days. One unknown minor degradate was formed with < 1.5% AR at any time point throughout the preliminary or confirmatory tests. Based on the findings, it was concluded that the substance is hydrolytically stable under acidic, neutral and alkaline conditions.

Description of key information

Hydrolytic DT50 at pH 7 at 25 °C > 1 year, hydrolytically stable under acidic, neutral, and basic conditions, OECD TG 111 and EPA 161 -1, Berdat and Nicollier 2007

Additional information

There is one hydrolysis study available, which followed OECD TG 111 and EPA 161-1 guideline and was in compliance with GLP criteria. In the preliminary test, sterile aqueous buffer solutions (pH 4, pH 5, pH 7 and pH 9) were prepared containing 0.32 mg/L 14C-pyrazole-labelled test substance. Each treated buffer was then incubated at 50°C in sealed vessels with no facility for trapping CO2 and organic volatiles for up to 5 days in the dark. Duplicate samples were taken daily for analysis during the incubation by LSC and HPLC. In a confirmatory test, the 14C-labelled test substance was incubated at pH 5, 7 and 9 at 25˚C for 30 days in the dark. Duplicate samples were taken on day 0, 15 and 30 during the test. In the preliminary test at 50 °C, the mean recoveries of radioactivity ranged from 90.9% to 99.3% of the applied radioactivity (AR) for all pH values. In the confirmatory test at 25 °C, the corresponding range was 91.2% to 99.3% AR. Less than 10% hydrolysis of test substance was observed for all four pH values after 5 days at 50 °C (equates to a DT50 > 1 year at 25 °C). The test substance was confirmed to be hydrolytically stable at pH 5, 7 and 9 at 25 ˚C for 30 days. Based on the findings, it was concluded that the substance is hydrolytically stable under acidic, neutral and alkaline conditions.