rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Sep 2014 to 05 Jan 2015

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:

adopted 13 Apr 2004

Deviations:

yes

Remarks:

No sterility confirmation tests were conducted at the end of the higher Tier test. Control of the 70°C hydrolysis vessels did not maintain 70 ± 0.5°C for the duration of the incubation period. This does not impact the integrity of the results.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Sampling intervals for the parent/transformation products:
- 70ºC: Triplicate samples: 0, 3, 7, 11, 16, 23 and 30 d.
- 50ºC: Triplicate samples: 0, 3, 7, 11, 16, 23 and 30 d.
- 25ºC: Triplicate samples: 0, 3, 7, 11, 16, 23 and 30 d.
- Sampling method: Complete treated samples were removed at each sampling time
- Sampling intervals/times for pH measurements: pH measured at each sampling point. At 70°C values ranged from pH 8.95 to 9.09. At 50ºC values ranged from pH 9.00 to 9.33. At 25ºC values ranged from pH 8.99 to 9.13.
- Sampling intervals/times for sterility check: No sterility confirmation tests were conducted at the end of the higher Tier test.
- Sample storage conditions before analysis: All samples quantified and analysed on the day of sampling

Buffers:

- pH: 4, 7 and 9
- Type and final molarity of buffer: 0.05 M
- Composition of buffer:
- pH 4: Ammonium Acetate prepared in Milli-Q Water and buffered with Formic Acid
- pH 7: Ammonium Hydrogen Carbonate prepared in Milli-Q Water and buffered with Formic Acid
- pH 9: Ammonium Hydrogen Carbonate prepared in Milli-Q Water and buffered with Ammonia (ca 32%).

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: Amber glass vessels of 50 mL capacity with ground glass lids
- Sterilisation method: Filtration (solutions), Autoclaving (vessels)
- Lighting: Dark
- Measures to exclude oxygen: Oxygen depleted by bubbling with nitrogen
- Details on test procedure for unstable compounds: No sterility confirmation tests were conducted at the end of the higher Tier test
- Details of traps for volatile, if any: Sealed vessel, no collection of volatiles
- Is there any indication of the test material adsorbing to the walls of the test apparatus? No

TEST MEDIUM
- Volume used/treatment: 40 mL
- Kind of water: Milli-Q water
- Identity and concentration of co-solvent: Acetonitrile, final concentration of 1.0% v/v in each test solution

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

ca. 0.15 mg/L

Remarks:

Values determined at 2.4 hours, 1 day and 5 days

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 0.15 mg/L

Remarks:

Values determined at 2.4 hours, 1 day and 5 days

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 0.15 mg/L

Remarks:

Values determined at 2.4 hours, 1 day and 5 days

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.148 mg/L

Remarks:

Values determined at 0, 3, 7, 11, 16, 23 and 30 days

Duration:

30 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.148 mg/L

Remarks:

Values determined at 0, 3, 7, 11, 16, 23 and 30 days

Duration:

30 d

pH:

9

Temp.:

70 °C

Initial conc. measured:

0.148 mg/L

Remarks:

Values determined at 0, 3, 7, 11, 16, 23 and 30 days

Number of replicates:

3

Positive controls:

no

Negative controls:

no

Preliminary study:

A hydrolysis rate screening test was carried out at 50 ± 0.5°C at pH 4, 7 and 9 in suitable buffered solutions (Tier 1).The decrease in concentration with time was followed by comparing the initial concentration with values determined at 2.4 hours, 1 day and 5 days. Assuming a pseudo first order hydrolysis, the rate constant for the reaction demonstrated that there had been no degradation of the test item in solutions buffered at pH 4 or pH 7. However from tests carried out in solutions buffered at pH 9 highlighted that there had been significant (>10%) degradation of the test item. As it was evident that > 10% of the test item had been hydrolysed at pH 9 over the 5 day testing, period additional (Tier 2) testing was required as detailed in OECD guideline 111.

Transformation products:

not measured

Remarks:

No significant hydrolysis products were observed at environmentally relevant temperatures

% Recovery:

84.6

St. dev.:

8.1

pH:

4

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

The hydrolysis of the sample was also measured at timepoints 0, 0.1 and 1

% Recovery:

95

St. dev.:

2.9

pH:

7

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

Remarks:

The hydrolysis of the sample was also measured at timepoints 0, 0.1 and 1

% Recovery:

75.8

St. dev.:

0.5

pH:

9

Temp.:

50 °C

Duration:

5 d

Remarks on result:

other: Hydrolysis more than 10%

Remarks:

The hydrolysis of the sample was also measured at timepoints 0, 0.1 and 1

% Recovery:

83.7

St. dev.:

14.3

pH:

9

Temp.:

25 °C

Duration:

30 d

Remarks on result:

other:

Remarks:

The hydrolysis of the sample was also measured at timepoints 0, 3,7,11,16 and 23.

% Recovery:

55.1

St. dev.:

8.9

pH:

9

Temp.:

50 °C

Duration:

30 d

Remarks on result:

other: Hydrolysis proportionally slower

Remarks:

The hydrolysis of the sample was also measured at timepoints 0, 3,7,11,16 and 23.

% Recovery:

< 3.4

pH:

9

Temp.:

70 °C

Duration:

30 d

Remarks on result:

other: Values

Remarks:

The hydrolysis of the sample was also measured at timepoints 0, 3,7,11,16 and 23 days

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.002 d-1

DT50:

384 d

Type:

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

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.014 d-1

DT50:

50 d

Type:

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

pH:

9

Temp.:

70 °C

Hydrolysis rate constant:

0.086 d-1

DT50:

8.1 d

Type:

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

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes, the temperatures remained constant throughout the incubation periods (25 ± 0.5°C, 50 ± 0.5°C and 70 ± 1.0 °C) and there was no significant variation in the pH values of the buffered solutions

Table 1. Hydrolysis of Samples Prepared in pH4 Buffer (Tier 1, 50°C)

Timepoint (Days)	Replicate	Sample Concentration (µg/mL)	Determined Concentration (µg/mL)	Recovery (%)	Ln[Recovery]	Mean Determined Recovery (%)	Coefficient of Variation (%)	Ln[Mean Recovery]
0	A B C	148.3	136.5 137.8 136.2	92.0 92.9 91.8	4.52 4.53 4.52	92.2	0.6	4.52
0.1	A B C	148.3	134.9 134.8 137.2	91.0 90.9 92.5	4.51 4.51 4.53	91.5	1.0	4.52
1	A B C	148.3	133.5 135.3 136.5	90.0 91.2 92.0	4.50 4.51 4.52	91.1	1.1	4.51
5	A B C	148.3	123.1 116.7 136.8	83.0 78.7 92.2	4.42 4.37 4.52	84.6	8.1	4.44

Table 2. Hydrolysis of Samples Prepared in pH7 Buffer (Tier 1, 50°C)

Timepoint (Days)	Replicate	Sample Concentration (µg/mL)	Determined Concentration (µg/mL)	Recovery (%)	Ln[Recovery]	Mean Determined Recovery (%)	Coefficient of Variation (%)	Ln[Mean Recovery]
0	A B C	148.3	131.2 128.8 129.4	88.5 86.9 87.3	4.48 4.46 4.47	87.6	1.0	4.47
0.1	A B C	148.3	136.3 135.9 135.9	91.9 91.6 91.6	4.52 4.52 4.52	91.7	0.2	4.52
1	A B C	148.3	138.9 138.8 136.1	93.7 93.6 91.8	4.54 4.54 4.52	93.0	1.1	4.53
5	A B C	148.3	138.5 138.6 145.7	93.4 93.5 98.2	4.54 4.54 4.59	95.0	2.9	4.55

Table 3. Hydrolysis of Samples Prepared in pH9 Buffer (Tier 1, 50°C)

Timepoint (Days)	Replicate	Sample Concentration (µg/mL)	Determined Concentration (µg/mL)	Recovery (%)	Ln[Recovery]	Mean Determined Recovery (%)	Coefficient of Variation (%)	Ln[Mean Recovery]
0	A B C	148.3	130.2 133.8 134.1	87.8 90.2 90.4	4.48 4.50 4.50	89.5	1.6	4.49
0.1	A B C	148.3	136.5 136.8 135.1	92.0 92.2 91.1	4.52 4.52 4.51	91.8	0.6	4.52
1	A B C	148.3	135.1 135.6 135.2	91.1 91.4 91.2	4.51 4.52 4.51	91.2	0.2	4.51
5	A B C	148.3	112.9 111.7 112.6	76.1 75.3 75.9	4.33 4.32 4.33	75.8	0.5	4.33

Table 4. Hydrolysis of Samples Prepared in pH9 Buffer (Tier 2, 25°C)

Timepoint (Days)	Replicate	Sample Concentration (µg/mL)	Determined Concentration (µg/mL)	Recovery (%)	Ln[Recovery]	Mean Determined Recovery (%)	Coefficient of Variation (%)	Ln[Mean Recovery]
0	A B C	148.3	136.0 130.9 131.6	91.7 88.3 88.7	4.52 4.48 4.49	89.6	2.1	4.50
3	A B C	148.3	135.1 138.7 136.1	91.1 93.5 91.8	4.51 4.54 4.52	92.1	1.3	4.52
7	A B C	148.3	119.6 119.2 122.7	80.6 80.4 82.7	4.39 4.39 4.42	81.2	1.6	4.40
11	A B C	148.3	132.0 134.8 137.5	89.0 90.9 92.7	4.49 4.51 4.53	90.9	2.0	4.51
16	A B C	148.3	137.7 133.3 137.8	92.9 89.9 92.9	4.53 4.50 4.53	91.9	1.9	4.52
23	A B C	148.3	119.4 128.5 132.0	80.5 86.6 89.0	4.39 4.46 4.49	85.4	5.1	4.45
30	A B C	148.3	103.7 134.0 134.6	69.9 90.4 90.8	4.25 4.50 4.51	83.7	14.3	4.43

Table 5. Hydrolysis of Samples Prepared in pH9 Buffer (Tier 2, 50°C)

Timepoint (Days)	Replicate	Sample Concentration (µg/mL)	Determined Concentration (µg/mL)	Recovery (%)	Ln[Recovery]	Mean Determined Recovery (%)	Coefficient of Variation (%)	Ln[Mean Recovery]
0	A B C	148.3	133.2 125.8 124.5	89.8 84.8 84.0	4.50 4.44 4.43	86.2	3.6	4.46
3	A B C	148.3	136.0 135.3 136.3	91.7 91.2 91.9	4.52 4.51 4.52	91.6	0.4	4.52
7	A B C	148.3	124.7 130.7 121.6	84.1 88.1 82.0	4.43 4.48 4.41	84.7	3.7	4.44
11	A B C	148.3	117.3 115.8 125.1	79.1 78.1 84.4	4.37 4.36 4.44	80.5	4.2	4.39
16	A B C	148.3	125.3 45.6* 126.3	84.5 N/C 85.2	4.44 N/C 4.45	84.9	0.6	4.44
23	A B C	148.3	104.0 120.1 104.7	70.1 81.0 70.6	4.25 4.39 4.26	73.9	8.3	4.30
30	A B C	148.3	75.9 79.4 89.8	51.2 53.5 60.6	3.94 3.98 4.10	55.1	8.9	4.01

Table 6. Hydrolysis of Samples Prepared in pH9 Buffer (Tier 2, 70°C)

Timepoint (Days)	Replicate	Sample Concentration (µg/mL)	Determined Concentration (µg/mL)	Recovery (%)	Ln[Recovery]	Mean Determined Recovery (%)	Coefficient of Variation (%)	Ln[Mean Recovery]
0	A B C	148.3	135.1 131.2 131.5	91.1 88.5 88.7	4.51 4.48 4.49	89.4	1.6	4.49
3	A B C	148.3	126.7 125.6 126.6	85.4 84.7 85.4	4.45 4.44 4.45	85.2	0.5	4.45
7	A B C	148.3	118.4 112.0 116.8	79.8 75.5 78.8	4.38 4.32 4.37	78.0	2.9	4.36
11	A B C	148.3	57.7 85.4 78.8	38.9 57.6 53.1	3.66 4.05 3.97	49.9	19.6	3.91
16	A B C	148.3	30.7 33.9 34.2	20.7 22.9 23.1	3.03 3.13 3.14	22.2	6.0	3.10
23	A B C	148.3	24.2 <LOQ* <LOQ*	16.3 <3.4* <3.4*	2.79 N/C N/C	N/C	N/C	N/C
30	A B C	148.3	<LOQ* <LOQ* <LOQ*	<3.4* <3.4* <3.4*	N/C N/C N/C	N/C	N/C	N/C

Validity criteria fulfilled:

not specified

Conclusions:

The test material was found hydrolytically stable under neutral (pH=7) or acidic (pH=4) conditions at environmental relevant temperatures, without significant degradation and hydrolysis products observed. Under basic conditions hydrolysis was detected at higher temperatures (i.e. 50°C and 70°C). The half-life of the test item at pH=9 at 25°C was 384d, with a pseudo-first-order rate constant of 0.001803 d-1.

Executive summary:

The hydrolytic stability of the test material was determined according to guideline OECD 111 under GLP. The hydrolysis of the test material at 0.15 mg/L was studied in sterilised aqueous buffered solutions at pH 4, 7 and 9 in the dark at 50 °C for 5 days. In each initial test, triplicate samples were taken for analysis at 4 intervals during incubation. After addition of an internal standard, the aqueous solutions were analysed for quantification purposes by HPLC-UV analysis. As greater than 10% degradation occurred at pH 9 during the initial investigation, further testing was conducted. The hydrolysis of the test material at 0.15 mg/L was studied in sterilised aqueous buffered solutions at pH 9 in the dark at 70 °C, 50 °C and 25 °C for up to 30 days. In each test, triplicate samples were taken for analysis at 7 intervals during incubation. After addition of an internal standard, the aqueous solutions were analysed for quantification purposes by HPLC-UV analysis. The stability of stock solutions of of the test material, prepared in acetonitrile, was confirmed for at least 32 days when stored at ca 4°C.

During initial testing the testing material appeared to degrade slowly at pH 9 and 50 ºC, but no degradation was observed at pH 4 or pH 7 at 50 ºC. After further testing at pH 9 it was demonstrated that the rate of degradation was proportionally slower at 50 ºC and 25 ºC than at 70 ºC. At 70 ºC, concentration of the parent compound decreased from 89.4% at day 0 to less than 3.4% of the initial after 30 days; at 50 ºC from 86.2% to 55.1% of the initial concentration after 30 days; and at 25 ºC from 89.6% to 83.7% of the initial concentration after 30 days. The Kobs (hydrolysis rate constant), half life and activation energy values were calculated using non-linear regression and pseudo first-order kinetics. At 25 ºC pH 9, no significant degradation was observed by HPLC-UV analysis and no significant hydrolysis product was observed. The temperatures remained constant throughout the incubation periods (25 ± 0.5°C, 50 ± 0.5°C and 70 ± 1.0 °C) and there was no significant variation in the pH values of the buffered solutions. The stability of the test material, in acetonitrile was confirmed for at least 32 days when stored at ca 4°C; mean recovery of freshly made standards from a stock solution stored at 4°C for 32 days compared to the actual concentration of freshly made standards from the stock solution freshly prepared was 97.4%.

This study demonstrated that the test material is hydrolytically stable under neutral or acidic conditions at environmental relevant temperatures. The test material was prone to hydrolysis under basic conditions, but only at elevated temperatures of 50 and 70 °C. At the environmentally relevant temperature of 25 °C no significant degradation and no significant hydrolysis products were observed. The half-life of the test item at pH=9 at 25°C was 384d, with a pseudo-first-order rate constant of 0.001803 d-1.

Description of key information

The test material was found hydrolytically stable under neutral or acidic conditions; under basic conditions hydrolysis was detected at higher temperatures (i.e. 50°C and 70°C). Half-life: 384d (pH=9, 25°C) (Clipston 2015)

Key value for chemical safety assessment

Half-life for hydrolysis:

384 d

at the temperature of:

25 °C

Additional information

During initial testing the testing material appeared to degrade slowly at pH 9 and 50 ºC, but no degradation was observed at pH 4 or pH 7 at 50 ºC. After further testing at pH 9 it was demonstrated that the rate of degradation was proportionally slower at 50 ºC and 25 ºC than at 70 ºC. At 70 ºC, concentration of the parent compound decreased from 89.4% at day 0 to less than 3.4% of the initial after 30 days; at 50 ºC from 86.2% to 55.1% of the initial concentration after 30 days; and at 25 ºC from 89.6% to 83.7% of the initial concentration after 30 days. The Kobs (hydrolysis rate constant), half life and activation energy values were calculated using non-linear regression and pseudo first-order kinetics. At 25 ºC pH 9, no significant degradation was observed by HPLC-UV analysis and no significant hydrolysis product was observed. The temperatures remained constant throughout the incubation periods (25 ± 0.5°C, 50 ± 0.5°C and 70 ± 1.0 °C) and there was no significant variation in the pH values of the buffered solutions.