1,2,3,4,4a,7,8,8a-octahydro-2,4a,5,8a-tetramethyl-1-naphthyl formate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 June 2014 and 06 October 2014.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Although a guideline study performed to GLP, the data at pH4 and 7 is confounded by non-pseudo first-order kinetics. Thus the study is considered reliable with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: SC00010054
Purity: 96.5%
Physical State / Appearance: Clear colorless liquid

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Duplicate vessels were removed for analysis at each timepoint.

Buffers:

These solutions were subjected to ultrasonication and degassing with nitrogen to minimise dissolved oxygen content. Buffers for preliminary testing were filtered through 0.2 µm filters before used, all other buffers were boiled and filtered through 0.2 µm filters before use.

Details on test conditions:

Preparation of the Test Solutions
Sample solutions were prepared in glass vessels at a nominal concentration of 1.0 mg/L in the three buffer solutions. A 1% co-solvent of acetonitrile was used to aid solubility.

The concentration of each solution did not exceed the lesser of 0.01 mol/L or half the water solubility.

The test solutions were split into individual vessels for each data point. Duplicate vessels were removed for analysis at each timepoint.

The solutions were shielded from light whilst maintained at the test temperature.

Preliminary Test
Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5 °C for a period of 96 hours, using unboiled filtered buffers (Preliminary Test 1). Preliminary tests were repeated with sample solutions at pH 4, 7 and 9 being maintained at 50.0 ± 0.5 °C for a period of 120 hours, using boiled filtered buffers (Preliminary Test 2).

Tier 2
Results from the preliminary tests showed it was necessary to undertake further testing at pH 4 and pH 7 with solutions being maintained at 25 ± 0.5 °C for a period of 35 days.
Results from the preliminary tests showed it was necessary to undertake further testing at pH 9, with solutions being maintained at 40 ± 0.5 °C for a period of 120 hours and 60 ± 0.5 °C for a period of 98 hours.
Testing was carried out at pH 4 with solutions being maintained at 40 ± 0.5 °C for a period of 120 hours and 60 ± 0.5 °C for a period of 98 hours.

Analysis of Sample Solutions
The sample solutions were taken from the waterbath at various times and the pH of each solution recorded.

The concentration of the sample solution was determined by gas chromatography (GC).

Samples
An aliquot (25 mL) of each sample solution was concentrated into 5 mL of hexane, by shaking with the sample. The sample was then allowed to separate. When separation had occurred, the hexane layer was vialed for analysis.

Standards
Duplicate standard solutions of test item were prepared in hexane at a nominal concentration of 5 mg/L.

Matrix blanks
An aliquot (25 mL) of relevant buffer solution was concentrated into 5 mL of by shaking, with the sample. The sample was then allowed to separate. When separation had occurred, the hexane layer was vialed for analysis.

Analysis
The standard and sample solutions were analysed by GC using the following conditions:
GC System: Agilent Technologies 6890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.25 mm id x 0.25 µm film)
Oven temperature program: initial 80 °C for 1 minute, rate 15 °C/min, final 300 °C for 5 minute
Injection temperature: 250 °C
FID detector temperature: 250 °C
Injection volume: 2 µL
Retention time: ~ 8.6 minutes

Calculations
The response factors of the standard peak areas (unit peak area per mg/L) were calculated using the equation below:

RF = (Rstd / Cstd)

Where:
RF = response factor for the standard solution
Rstd = peak area for the standard solution
Cstd = concentration for the standard solution (mg/L)

Testing the reaction for pseudo first order
The general rate expression for a first order reaction is:

d(c) / dt = - k c

or in the integrated form:

-log(c0 / ct) = -k •t

or

logcr = (-k) • t-logc0 (equation of a linear function)

Therefore hte rate constand k is the slope of a plot of log ct versus t. The data were plotted as log ct versus t. The linear plots prove that the hydrolysis reaction is pseudo first order. The reaction rate constant k can be calculated by regression analysis or the following equation:

-k = -(1/t) • log (c0 / ct)

The half life time was calculated from the following equation:

t0.5 = -0.693 / k

Where:
c0 = concentration of the test solution at time 0 [g/L]
ct = concentration of the test solution at time t [g / L]
k = first order rate constant [hours-1]
t0.5 = half life time [hours]

Evaluation of the rate constant at 25 °C
When the rate constants are known for two or more temperatures, the rate constants at other temperatures can be calculated using the Arrhenius equation:

k = A•e -(E / R•T)

Or

1n k = -(E/R)•(1/T)+ 1n A

A plot of ln k versus 1/T gives a straight line with a slope of -E/R

Where:

k = Rate constant, measured at different temperatures
E = Activation energy [kJ/mol]
T = Absolute temperature [K]
R = Gas constant [8.314 J/mol K]
A = Pre-exponential factor

The half life at 25 °C was extrapolated directly from the graph.

Duration:

35 d

pH:

4

Temp.:

25 °C

Remarks:

final test

Duration:

35 d

pH:

7

Temp.:

25 °C

Remarks:

final test

Duration:

120 h

pH:

9

Temp.:

40 °C

Remarks:

Tier 2 test

Duration:

96 h

pH:

9

Temp.:

50 °C

Remarks:

Preliminary test 2

Duration:

98 h

pH:

9

Temp.:

60 °C

Remarks:

Tier 2 test

Preliminary study:

pH 4, 7 and 9 at 50.0 ± 0.5 °C - Preliminary Test 1
The results from Preliminary Test 1 at 50.0 ± 0.5 °C showed a strong second order correlation after 96 hours. Therefore further preliminary testing was carried out 50 ± 0.5 °C (Preliminary Test 2), using boiled and filtered buffers as an additional precaution against any possible residual dissolved oxygen.

pH 4 & 7 at 50.0 ± 0.5 ºC – Preliminary Test 2
In the absence of any significant correlation/trend, but failure to analytically confirm stability through demonstrating less than 10% hydrolysis after 120 hours incubation at 50 ± 0.5°C further testing was required. A further test (Tier 2) was conducted at 25.0 ± 0.5 °C over an extended period of time to establish if hydrolysis was occurring, and if so, at which order. In addition further testing (Tier 2) was performed at 40.0 ± 0.5 °C over 120 hours and 60.0 ± 0.5 °C over 98 hours to further evaluate if second order kinetics were occurring.

pH 9 at 50.0 ± 0.5 ºC – Preliminary Test 2
The extent of hydrolysis after 120 hours indicated that further tests (Tier 2), conducted at 40.0 °C and 60.0 °C, were required to estimate the rate constant and half-life.

Slope = -1.19E-02
kob
= 2.74E-02 hour-1
= 7.61E-06 second-1
t½ = 25.3 hours

Transformation products:

yes

No.:

#1

No.:

#2

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

Identification of Hydrolysis Products
Degradant products were seen during the hydrolysis at pH 9 at 40 °C, 50 °C and 60 °C. The retention times for the degradant products are reported in the text of the study report as approximately 7.7 and 8.3 mins but from visual inspection of the typical chromatogram included in the report (page 36) are approximately 7.8 and 7.9 minutes compared to 8.86 for the parent substance). These have been provisionally attributed to the base catalysed hydrolysis of the ester functional group and the formation of corresponding alcohol and formic acid. Based on the relatively size of the two new peaks, the major peak at 7.9 is attributed to 2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphalen-1-ol (hydrolysis product of the mono-constituent component) and the minor peak at 7.8 to 2,5,5,8a-tetramethyl-1,2,3,5,6,7,8,8a--octahydronaphalen-1-ol (hydrolysis product of the isomer impurity). The formic acid released is unlikely to be observed by GC, eluting probably with the large solvent peak at retention time 1-2 minutes.

% Recovery:

94

pH:

4

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: mean of the two replicates

% Recovery:

90

pH:

7

Temp.:

25 °C

Duration:

35 d

Remarks on result:

other: mean of the two replicates

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

368 h

Type:

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

Details on results:

Tier 2
pH 4 at 25.0 ± 0.5 ºC showed no significant hydrolysis.
pH 4 at 40.0 ± 0.5 ºC
Slope = -3.57E-03
kobs
= 8.22E-03 hour-1
= 2.28E-06 second-1
t½ = 84.3 hours
pH 4 at 60.0 ± 0.5 ºC showed second order kinetics.
pH 7 at 25.0 ± 0.5 ºC showed no significant hydrolysis
pH 9 at 40.0 ± 0.5 ºC showed second order kinetics.
pH 9 at 60.0 ± 0.5 ºC
Slope = -2.59 E-02
kobs
= 5.96E-02 hour-1
= 1.66E-05 second-1
t½ =11.6 hours

The Arrhenius plot was constructed (pH 9). The rate constant and half-life at 25 °C have been estimated to be as follows:
kobs
= 1.88E-03 hour-1
= 5.23E-07 second-1
t½ = 368 hours

Validation
The linearity of the detector response with respect to concentration was assessed over the nominal concentration range of 0.2 to 10 mg/L. This was satisfactory with a correlation coefficient (r) of 0.999 being obtained. This reflects the linearity of the GC-system within the calibration range of 0.209 mg/L to 2.09 mg/L of test item within the initial sample solutions.

Recovery of analysis of the sample procedure was assessed for each pH and proved adequate for the test at a nominal concentration of 1 mg/L, recovery data is shown in the following table:

A nominal concentration of 1 mg/L at pH 4 gave a recovery range of 94.1 to 98.7 % (mean recovery 96.9 %).
A nominal concentration of 1 mg/L at pH 7 gave a recovery range of 96.4 to 98.7 % (mean recovery 98 %).
A nominal concentration of 1 mg/L at pH 9 gave a recovery range of 90.9 to 95.6 % (mean recovery 93.1 %).
Concentrations have not been corrected for recovery of analysis.

Discussion
The kinetics of the hydrolysis at pH 9 has been determined to be consistent with that of a pseudo-first order reaction as the graphs of log10 concentration versus time are straight lines.

No significant peaks were observed at the approximate retention time of the test item on analysis of any matrix blank solutions.

It has been observed that the rate of hydrolysis increases with an increase in pH.

The preliminary hydrolysis testing for pH 4 and pH 7 was repeated a number of times as the degradation was consistent with second order kinetics and therefore not considered due to hydrolysis alone. Even after removal of any possible residual oxygen by boiling the buffers and purging with nitrogen, second order kinetics were still observe. An attempt was made to perform testing at an varying temperatures (40 ± 0.5 °C and 60 ± 0.5 °C) at pH 4, again non-pseudo first order kinetics were observed. It was anticipated that testing at pH 7 would yield similar results.
It was therefore decided to perform testing directly at 25 ± 0.5 °C for a period of 30 days. Although testing continued into the 35th day, this was considered insignificant based on the fact that approximately 10% hydrolysis was observed for both pH 4 and pH 7.

Preliminary tests

The mean peak areas relating to the standard and sample solutions are shown in the following tables:

pH 4 at 50.0 ± 0.5 °C – Preliminary Test 1

Solution	Mean Peak Area
Standard 5.01 mg/L	1.2846 x 10^5
Standard 5.06 mg/L	1.3017 x 10^5
Initial Sample A, pH 4	1.2196 x 10^5
Initial Sample B, pH 4	1.2337 x 10^5
2 Hour Sample A, pH 4	1.0166 x 10^5
2 Hour Sample B, pH 4	9.8091 x 10^4
4 Hour Sample A, pH 4	9.0210 x 10^4
4 Hour Sample B, pH 4	9.1426 x 10^4
Standard 5.01 mg/L	1.2337 x 10^5
Standard 5.02 mg/L	1.2211 x 10^5
24 Hour Sample A, pH 4	4.2638 x 10^4
24 Hour Sample B, pH 4	3.8377 x 10^4
Standard 5.13 mg/L	1.2934 x 10^5
Standard 5.04 mg/L	1.2791 x 10^5
49 Hour Sample A, pH 4	3.4261 x 10^4
49 Hour Sample B, pH 4	3.0812 x 10^4
Standard 5.07 mg/L	1.2020 x 10^5
Standard 5.03 mg/L	1.2636 x 10^5
72.5 Hour Sample A, pH 4	2.3809 x 10^4
72.5 Hour Sample B, pH 4	2.1409 x 10^4
Standard 5.05 mg/L	1.2572 x 10^5
Standard 5.01 mg/L	1.2094 x 10^5
96 Hour Sample A, pH 4	1.6867 x 10^4
96 Hour Sample B, pH 4	2.0749 x 10^4

pH 7 at 50.0 ± 0.5 °C – Preliminary Test 1

Solution	Mean Peak Area
Standard 5.01 mg/L	1.2665 x 10^5
Standard 5.06 mg/L	1.2911 x 10^5
Initial Sample A, pH 7	1.1932 x 10^5
Initial Sample B, pH 7	1.1882 x 10^5
2 Hour Sample A, pH 7	9.1535 x 10^4
2 Hour Sample B, pH 7	9.7966 x 10^4
4 Hour Sample A, pH 7	8.2517 x 10^4
4 Hour Sample B, pH 7	8.2051 x 10^4
Standard 5.01 mg/L	1.2337 x 10^5
Standard 5.02 mg/L	1.2211 x 10^5
24 Hour Sample A, pH 7	4.1319 x 10^4
24 Hour Sample B, pH 7	4.6819 x 10^4
Standard 5.13 mg/L	1.2909 x 10^5
Standard 5.04 mg/L	1.2686 x 10^5
49 Hour Sample A, pH 7	3.4182 x 10^4
49 Hour Sample B, pH 7	3.0697 x 10^4
Standard 5.07 mg/L	1.1816 x 10^5
Standard 5.03 mg/L	1.2683 x 10^5
72.5 Hour Sample A, pH 7	2.3245 x 10^4
72.5 Hour Sample B, pH 7	2.0779 x 10^4
Standard 5.05 mg/L	1.2594 x 10^5
Standard 5.01 mg/L	1.2095 x 10^5
96 Hour Sample A, pH 7	1.6619 x 10^4
96 Hour Sample B, pH 7	1.8634 x 10^4

pH 9 at 50.0 ± 0.5 °C – Preliminary Test 1

Solution	Mean Peak Area
Standard 5.01 mg/L	1.2538 x 10^5
Standard 5.06 mg/L	1.2784 x 10^5
Initial Sample A, pH 9	1.1852 x 10^5
Initial Sample B, pH 9	1.1629 x 10^5
2 Hour Sample A, pH 9	9.2638 x 10^4
2 Hour Sample B, pH 9	8.8546 x 10^4
4 Hour Sample A, pH 9	7.5832 x 10^4
4 Hour Sample B, pH 9	7.5114 x 10^4
Standard 5.01 mg/L	1.2337 x 10^5
Standard 5.02 mg/L	1.2211 x 10^5
24 Hour Sample A, pH 9	2.3531 x 10^4
24 Hour Sample B, pH 9	2.6931 x 10^4
Standard 5.13 mg/L	1.2826 x 10^5
Standard 5.04 mg/L	1.2665 x 10^5
49 Hour Sample A, pH 9	1.1175 x 10^4
49 Hour Sample B, pH 9	1.0294 x 10^4
Standard 5.07 mg/L	1.1753 x 10^5
Standard 5.03 mg/L	1.2705 x 10^5
72.5 Hour Sample A, pH 9	6.4365 x 10^3
72.5 Hour Sample B, pH 9	6.4345 x 10^3
Standard 5.05 mg/L	1.2571 x 10^5
Standard 5.01 mg/L	1.2108 x 10^5
96 Hour Sample A, pH 9	4.5690 x 10^3
96 Hour Sample B, pH 9	4.8495 x 10^3

The test item concentrations at the given time points are shown in the following tables:

pH 4 at 50.0 ± 0.5 ºC – Preliminary Test 1

Time (Hours)	Concentration (g/L)	Log Concentration	% mean initial concentration
0 (A)	9.49 x 10^-4	-3.02	-
0 (B)	9.60 x 10^-4	-3.02	-
2 (A)	7.91 x 10^-4	-3.10	82.9
2 (B)	7.64 x 10^-4	-3.12	80.0
4 (A)	7.02 x 10^-4	-3.15	73.5
4 (B)	7.12 x 10^-4	-3.15	74.5
24 (A)	3.48 x 10^-4	-3.46	36.5
24 (B)	3.14 x 10^-4	-3.50	32.8
49 (A)	2.71 x 10^-4	-3.57	28.4
49 (B)	2.44 x 10^-4	-3.61	25.5
72.5 (A)	1.95 x 10^-4	-3.71	20.4
72.5 (B)	1.75 x 10^-4	-3.76	18.4
96 (A)	1.38 x 10^-4	-3.86	14.4
96 (B)	1.69 x 10^-4	-3.77	17.7

pH 7 at 50.0 ± 0.5 ºC – Preliminary Test 1

Time (Hours)	Concentration (g/L)	Log Concentration	% mean initial concentration
0 (A)	9.39 x 10^-4	-3.03	-
0 (B)	9.35 x 10^-4	-3.03	-
2 (A)	7.21 x 10^-4	-3.14	76.9
2 (B)	7.71 x 10^-4	-3.11	82.3
4 (A)	6.50 x 10^-4	-3.19	69.3
4 (B)	6.46 x 10^-4	-3.19	68.9
24 (A)	3.38 x 10^-4	-3.47	36.0
24 (B)	3.82 x 10^-4	-3.42	40.8
49 (A)	2.72 x 10^-4	-3.57	29.0
49 (B)	2.44 x 10^-4	-3.61	26.0
72.5 (A)	1.91 x 10^-4	-3.72	20.4
72.5 (B)	1.71 x 10^-4	-3.77	18.3
96 (A)	1.35 x 10^-4	-3.87	14.5
96 (B)	1.52 x 10^-4	-3.82	16.2

pH 9 at 50.0 ± 0.5 ºC – Preliminary Test 1

Time (Hours)	Concentration (g/L)	Log Concentration	% mean initial concentration
0 (A)	9.42 x 10^-4	-3.03	-
0 (B)	9.24 x 10^-4	-3.03	-
2 (A)	7.36 x 10^-4	-3.13	78.9
2 (B)	7.04 x 10^-4	-3.15	75.4
4 (A)	6.03 x 10^-4	-3.22	64.6
4 (B)	5.97 x 10^-4	-3.22	64.0
24 (A)	1.92 x 10^-4	-3.72	20.6
24 (B)	2.20 x 10^-4	-3.66	23.6
49 (A)	8.92 x 10^-5	-4.05	9.55
49 (B)	8.21 x 10^-5	-4.09	8.80
72.5 (A)	5.31 x 10^-5	-4.28	5.69
72.5 (B)	5.31 x 10^-5	-4.28	5.69
96 (A)	3.73 x 10^-5	-4.43	3.99
96 (B)	3.95 x 10^-5	-4.40	4.24

pH 4 at 50.0 ± 0.5 °C – Preliminary Test 2

Solution	Mean Peak Area
Standard 5.04 mg/L	1.2017 x 10^5
Standard 5.10 mg/L	1.2198 x 10^5
Initial Sample A, pH 4	1.2038 x 10^5
Initial Sample B, pH 4	1.2185 x 10^5
2 Hour Sample A, pH 4	1.1661 x 10^5
2 Hour Sample B, pH 4	1.1868 x 10^5
4 Hour Sample A, pH 4	1.0890 x 10^5
4 Hour Sample B, pH 4	1.0894 x 10^5
Standard 5.07 mg/L	1.2448 x 10^5
Standard 5.05 mg/L	1.2480 x 10^5
24 Hour Sample A, pH 4	1.0122 x 10^5
24 Hour Sample B, pH 4	1.0804 x 10^5
Standard 5.06 mg/L	1.2464 x 10^5
Standard 5.06 mg/L	1.2024 x 10^5
48 Hour Sample A, pH 4	1.1674 x 10^5
48 Hour Sample B, pH 4	1.1571 x 10^5
Standard 5.06 mg/L	1.2144 x 10^5
Standard 5.06 mg/L	1.1942 x 10^5
120 Hour Sample A, pH 4	1.0330 x 10^5
120 Hour Sample B, pH 4	9.5939 x 10^4

pH 7 at 50.0 ± 0.5 °C – Preliminary Test 2

Solution	Mean Peak Area
Standard 5.04 mg/L	1.1884 x 10^5
Standard 5.10 mg/L	1.2121 x 10^5
Initial Sample A, pH 7	1.1230 x 10^5
Initial Sample B, pH 7	1.1315 x 10^5
2 Hour Sample A, pH 7	1.0114 x 10^5
2 Hour Sample B, pH 7	9.9176 x 10^4
4 Hour Sample A, pH 7	1.0373 x 10^5
4 Hour Sample B, pH 7	1.0212 x 10^5
Standard 5.07 mg/L	1.2448 x 10^5
Standard 5.05 mg/L	1.2480 x 10^5
24 Hour Sample A, pH 7	1.0492 x 10^5
24 Hour Sample B, pH 7	1.0437 x 10^5
Standard 5.06 mg/L	1.2464 x 10^5
Standard 5.06 mg/L	1.2024 x 10^5
48 Hour Sample A, pH 7	1.1206 x 10^5
48 Hour Sample B, pH 7	1.1440 x 10^5
Standard 5.06 mg/L	1.2144 x 10^5
Standard 5.06 mg/L	1.1942 x 10^5
120 Hour Sample A, pH 7	9.3008 x 10^4
120 Hour Sample B, pH 7	9.1192 x 10^4

pH 9 at 50.0 ± 0.5 °C – Preliminary Test 2

Solution	Mean Peak Area
Standard 5.04 mg/L	1.1868 x 10^5
Standard 5.10 mg/L	1.2086 x 10^5
Initial Sample A, pH 9	1.0849 x 10^5
Initial Sample B, pH 9	1.1009 x 10^5
2 Hour Sample A, pH 9	8.9439 x 10^4
2 Hour Sample B, pH 9	8.8778 x 10^4
4 Hour Sample A, pH 9	8.8920 x 10^4
4 Hour Sample B, pH 9	8.4296 x 10^4
Standard 5.07 mg/L	1.2448 x 10^5
Standard 5.05 mg/L	1.2480 x 10^5
24 Hour Sample A, pH 9	4.8453 x 10^4
24 Hour Sample B, pH 9	5.0116 x 10^4
Standard 5.06 mg/L	1.2464 x 10^5
Standard 5.06 mg/L	1.2024 x 10^5
48 Hour Sample A, pH 9	2.9130 x 10^4
48 Hour Sample B, pH 9	2.8928 x 10^4
Standard 5.06 mg/L	1.2144 x 10^5
Standard 5.06 mg/L	1.1942 x 10^5
120 Hour Sample A, pH 9	3.5525 x 10^3
120 Hour Sample B, pH 9	3.8535 x 10^3

The test item concentrations at the given time points are shown in the following tables:

pH 4 at 50.0 ± 0.5 ºC – Preliminary Test 2

Time (Hours)	Concentration (g/L)	Log concentration	% mean initial concentration
0 (A)	1.01 x 10^‑3	-3.00	-
0 (B)	1.02 x 10^-3	-2.99	-
2 (A)	9.77 x 10^-4	-3.01	96.3
2 (B)	9.94 x 10^-4	-3.00	98.0
4 (A)	9.12 x 10^-4	-3.04	89.9
4 (B)	9.12 x 10^-4	-3.04	89.9
24 (A)	8.21 x 10^-4	-3.09	81.0
24 (B)	8.77 x 10^-4	-3.06	86.4
48 (A)	9.65 x 10^-4	-3.02	95.1
48 (B)	9.56 x 10^-4	-3.02	94.3
120 (A)	8.68 x 10^-4	-3.06	85.6
120 (B)	8.06 x 10^-4	-3.09	79.5

pH 7 at 50.0 ± 0.5 ºC – Preliminary Test 2

Time (Hours)	Concentration (g/L)	Log Concentration	% mean initial concentration
0 (A)	9.49 x 10^-4	-3.02	-
0 (B)	9.56 x 10^-4	-3.02	-
2 (A)	8.55 x 10^-4	-3.07	89.7
2 (B)	8.38 x 10^-4	-3.08	88.0
4 (A)	8.76 x 10^-4	-3.06	92.0
4 (B)	8.63 x 10^-4	-3.06	90.6
24 (A)	8.51 x 10^-4	-3.07	89.4
24 (B)	8.47 x 10^-4	-3.07	88.9
48 (A)	9.26 x 10^-4	-3.03	97.3
48 (B)	9.46 x 10^-4	-3.02	99.3
120 (A)	7.82 x 10^-4	-3.11	82.1
120 (B)	7.66 x 10^-4	-3.12	80.5

pH 9 at 50.0 ± 0.5 ºC – Preliminary Test 2

Time (Hours)	Concentration (g/L)	Log Concentration	% mean initial concentration
0 (A)	9.19 x 10^-4	-3.04	-
0 (B)	9.32 x 10^-4	-3.03	-
2 (A)	7.57 x 10^-4	-3.12	81.8
2 (B)	7.52 x 10^-4	-3.12	81.2
4 (A)	7.53 x 10^-4	-3.12	81.4
4 (B)	7.14 x 10^-4	-3.15	77.1
24 (A)	3.93 x 10^-4	-3.41	42.5
24 (B)	4.07 x 10^-4	-3.39	44.0
48 (A)	2.41 x 10^-4	-3.62	26.0
48 (B)	2.39 x 10^-4	-3.62	25.8
120 (A)	2.99 x 10^-4	-4.53	3.22
120 (B)	3.24 x 10^-4	-4.49	3.50

Validity criteria fulfilled:

yes

Remarks:

Recoveries of a standard 1mg/L samples were 97% (pH4), 98% (pH7) and 91% (pH9) and thus meet the OCED guidline qaulity criteria od 90 to 110% recoveries.

Conclusions:

The hydrolysis results at 25 °C showed that at pH 4, approximately 10 % hydrolysis after 35 days was observed.
The hydrolysis results at 25 °C showed that at pH 7, approximately 10 % hydrolysis after 35 days was observed.
The hydrolysis results at 25 °C showed that at pH 9, the rate constant was 5.23E-07 s-1 with a half life of 368 hours.

Executive summary:

Hydrolysis as a function of pH was assessed using a procedure in accordance with Method C7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004. Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5 °C for a period of 96 hours, using unboiled filtered buffers (Preliminary Test 1). The level of parent remaining after 96 hours was approximately 16% (pH4), 15% (pH7) and 4% (pH9). The data showed second order kinetics indicating that degradation was not due to hydrolysis alone. Therefore the preliminary tests were repeated using boiled and filtered buffers as an additional precaution against any possible residual dissolved oxygen for a period of 120 hours.

In preliminary test 2, no significant trend of concentration over time was observed at pH 4 and 7. However, failure to analytically confirm stability meant that < 10% hydrolysis could not be demonstrated. Therefore an attempt was made to perform testing at varying temperatures. In contrast to the preliminary test 2 at 50°C, a clear trend of decreasing concentration of the test item over time was observed with the % of parent remaining after 96 hours being approximately 37% (at 40°C) and 11% (at 60°C). Again, non-pseudo first order kinetics was observed. It was anticipated that testing at pH7 would yield similar results; i.e. that first-order hydrolysis would not be observed. It was therefore decided to perform testing directly at 25 ± 0.5 °C for a period of 30 days. Testing was continued into the 35th day, hydrolysis was considered insignificant based on the fact that only approximately 10% hydrolysis was observed for both pH 4 and pH 7.

The kinetics of the hydrolysis at pH 9 in preliminary test 2 (50°C) and two further tests (40 and 60°C) were consistent with that of a pseudo-first order reaction as the graphs of log10concentration versus time are straight lines. Therefore, the rate constant and half-life time of the test item at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25°C. The latter was calculated to be 368 hours.

Degradant products were seen during the hydrolysis at pH 9 at 40 °C, 50 °C and 60 °C. Two new peaks were observed at retention times of approximately 7.8 and 7.9 mins compared to 8.86 for the parent substance). These have been provisionally attributed to the base catalysed hydrolysis of the ester functional group and the formation of corresponding alcohol and formic acid. Based on the relatively size of the two new peaks, the major peak at 7.9 is attributed to 2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphalen-1-ol (hydrolysis product of the mono-constituent component) and the minor peak at 7.8 to 2,5,5,8a-tetramethyl-1,2,3,5,6,7,8,8a--octahydronaphalen-1-ol (hydrolysis product of the isomer impurity).

Description of key information

The test item was found to be hydrolytically unstable at pH 9 (t1/2 = 368 hours at 25°C, calculated using the Arrhenius equation from data obtained at 40, 50 and 60°C). Approximately 10% hydrolysis was observed at tests performed directly at 25°C at pH4 and pH7. This is considered insignificant and thus a worst case half-life of 1 year has been used as the key value for chemical safety assessment. Some degradation was observed at pH4 and pH7 at higher temperatures. However, non-pseudo first order kinetics indicate that this is not due to hydrolysis alone.

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

Hydrolysis as a function of pH was assessed using a procedure in accordance with Method C7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004. Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5 °C for a period of 96 hours, using unboiled filtered buffers (Preliminary Test 1). The level of parent remaining after 96 hours was approximately 16% (pH4), 15% (pH7) and 4% (pH9). The data showed second order kinetics indicating that degradation was not due to hydrolysis alone. Therefore the preliminary tests were repeated using boiled and filtered buffers as an additional precaution against any possible residual dissolved oxygen for a period of 120 hours.

In preliminary test 2, no significant trend of concentration over time was observed at pH 4 and 7. However, failure to analytically confirm stability meant that < 10% hydrolysis could not be demonstrated. Therefore an attempt was made to perform testing at varying temperatures. In contrast to the preliminary test 2, a clear trend of decreasing concentration of the test item over time was observed with the % of parent remaining after 96 hours being approximately 37% (at 40°C) and 11% (at 60°C). Again, non-pseudo first order kinetics was observed. It was anticipated that testing at pH7 would yield similar results; i.e. that first-order hydrolysis would not be observed. It was therefore decided to perform testing directly at 25 ± 0.5 °C for a period of 30 days. Testing was continued into the 35th day, hydrolysis was considered insignificant based on the fact that only approximately 10% hydrolysis was observed for both pH 4 and pH 7.

The kinetics of the hydrolysis at pH 9 in preliminary test 2 (50°C) and two further tests (40 and 60°C) were consistent with that of a pseudo-first order reaction as the graphs of log10 concentration versus time are straight lines. Therefore, the rate constant and half-life time of the test item at each temperature was obtained and the Arrhenius equation was used to determine the rate constant and half-life time at 25°C. The latter was calculated to be 368 hours.

Degradant products were seen during the hydrolysis at pH 9 at 40 °C, 50 °C and 60 °C. Two new peaks were observed at retention times of approximately 7.8 and 7.9 mins compared to 8.86 for the parent substance). These have been provisionally attributed to the base catalysed hydrolysis of the ester functional group and the formation of corresponding alcohol and formic acid. Based on the relatively size of the two new peaks, the major peak at 7.9 is attributed to 2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphalen-1-ol (hydrolysis product of the mono-constituent component) and the minor peak at 7.8 to 2,5,5,8a-tetramethyl-1,2,3,5,6,7,8,8a-octahydronaphalen-1-ol (hydrolysis product of the isomer impurity).