[No public or meaningful name is available]

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th August 2020 to 2nd February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Molecular formula:

C20H36O2

IUPAC Name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Test material form:

liquid: viscous

Details on test material:

Chemical name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1‐yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers

Specific details on test material used for the study:

Test Item

Identification: FRET 18-0091

Chemical name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1‐yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers

Appearance: light yellow viscous liquid

Batch: RDEA571-42

Purity: 92.0%

Expiry date: 01 December 2021

Storage conditions: room temperature, in the dark

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Remarks:

The concentration of test item in the sample solutions was determined by gas chromatography (GC).

Details on sampling:

Duplicate sample solutions were taken initially and from the waterbath at various time points. The pH of each solution was recorded.

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

Buffers:

The test system consisted of buffer solutions at pH’s 4, 7 and 9, detailed in the following table: (Please see 'Any other information on materials and methods incl. tables')

The buffer solutions were passed through a 0.2 µm membrane filter to sterilize and subjected to ultrasonication and degassing with nitrogen to minimize dissolved oxygen.

Details on test conditions:

Preliminary Test/Tier 1
A spiking solution was prepared at a nominal 50 mg/L in methanol. This was used to prepare stock solutions of test item at a nominal 0.5 mg/L in the three buffer solutions containing 1% of the acetonitrile as a co-solvent. The stock solutions were split into individual glass vessels, sealed with no headspace, for each data point. These sample solutions were shielded from light whilst maintained at the test temperature. The sample solutions were maintained at 50.0 ± 0.5 °C for a period of 5 days.

Preliminary Test/Tier 1 – Repeat
A spiking solution was prepared at a nominal 10 mg/L in acetone. This was used to prepare stock solutions of test item at a nominal 0.5 mg/L in the three buffer solutions containing 5% of the acetone as a co-solvent. The stock solutions were split into individual glass vessels, sealed with minimal headspace (nitrogen filled), for each data point. These sample solutions were shielded from light whilst maintained at the test temperature. The sample solutions were maintained at 50.0 ± 0.5 °C for a period of 5 days.

Tier 2
A spiking solution was prepared at a nominal 10 mg/L in acetone. This was used to prepare stock solutions of test item at a nominal 0.5 mg/L in the three buffer solutions containing 5% of the acetone as a co-solvent. The stock solutions were split into individual glass vessels, sealed with minimal headspace (nitrogen filled), for each data point. These sample solutions were shielded from light whilst maintained at the test temperature. The sample solutions were maintained at 25.0 ± 0.5 °C for a period of 28 days.

Duration of test

Duration of test 1

Duration:

120 h

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.408 - < 0.444 mg/L

Duration of test 2

Duration:

120 h

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.353 - < 0.404 mg/L

Remarks:

Tier 2

Duration of test 3

Duration:

120 h

pH:

4

Temp.:

25 °C

Initial conc. measured:

0.409 - < 0.415 mg/L

Remarks:

Tier 2

Number of replicates:

Due to an unexpected significant reduction in the concentration of the test item at all pHs, preliminary/tier 1 was repeated to investigate the possible loss of test item due to volatilizing and absorption to the vessel lid.

6 replicates per pH test (3 of sample A, 3 of sample B)

Positive controls:

no

Negative controls:

no

Results and discussion

Preliminary study:

Due to the nature/quantity of this information please see 'Any other information on results and discussion incl. tables'

Transformation products:

no

Total recovery of test substance (in %)

Total recovery of test substance (in %) 1

% Recovery:

94.9 - < 101

pH:

9

Temp.:

50 °C

Duration:

120 h

Total recovery of test substance (in %) 2

% Recovery:

94.4 - < 100

pH:

7

Temp.:

50 °C

Duration:

120 h

Total recovery of test substance (in %) 3

% Recovery:

91.4 - < 98.6

pH:

4

Temp.:

50 °C

Duration:

120 h

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

Dissipation DT50 of parent compound 2

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

Dissipation DT50 of parent compound 3

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

ca. 0 h-1

Any other information on results incl. tables

Preliminary Test/Tier 1

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

Table 2

Solution	Mean Peak Area
Standard 5.01 mg/L	2.6082 x 10^5
Standard 5.21 mg/L	2.7291 x 10^5
Initial Sample A, pH 4	2.2304 x 10^5
Initial Sample B, pH 4	2.1103 x 10^5
Initial Sample A, pH 7	2.2903 x 10^5
Initial Sample B, pH 7	2.4362 x 10^5
Initial Sample A, pH 9	2.3389 x 10^5
Initial Sample B, pH 9	2.3411 x 10^5
Standard 5.09 mg/L	3.6136 x 10^5
Standard 5.18 mg/L	3.6471 x 10^5
24 Hour Sample A, pH 4	8.7901 x 10^4
24 Hour Sample B, pH 4	8.1342 x 10^4
24 Hour Sample A, pH 7	9.8240 x 10^4
24 Hour Sample B, pH 7	1.3558 x 10^5
24 Hour Sample A, pH 9	1.1140 x 10^5
24 Hour Sample B, pH 9	1.0598 x 10^5
Standard 5.09 mg/L	3.3106 x 10^5
Standard 5.20 mg/L	3.4475 x 10^5
120 Hour Sample A, pH 4	1.9267 x 10^4
120 Hour Sample B, pH 4	1.9738 x 10^4
120 Hour Sample A, pH 7	2.1009 x 10^4
120 Hour Sample B, pH 7	2.3731 x 10^4
120 Hour Sample A, pH 9	2.1988 x 10^4
120 Hour Sample B, pH 9	2.5429 x 10^4

 

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

Table 3             pH 4 at 50 ºC

Time (Hours)	Concentration (mg/L)		% of Mean Initial Concentration
	A	B	A	B
0	0.427	0.404	103	97.2
24	0.124	0.115	29.9	27.7
120	0.029	0.030	7.06	7.24

Table 4             pH 7 at 50 ºC

Time (Hours)	Concentration (mg/L)		% of Mean Initial Concentration
	A	B	A	B
0	0.438	0.466	96.9	103
24	0.139	0.192	30.7	42.4
120	0.032	0.036	7.07	7.99

Table 5             pH 9 at 50 ºC

Time (Hours)	Concentration (mg/L)		% of Mean Initial Concentration
	A	B	A	B
0	0.448	0.448	100	100
24	0.157	0.150	35.2	33.5
120	0.033	0.039	7.48	8.65

Due to an unexpected significant reduction in the concentration of the test item at all pHs, preliminary/tier 1 was repeated to investigate the possible loss of test item due to volatilizing and absorption to the vessel lids.

 

 Preliminary Test/Tier 1 - Repeat

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

Table 6

Solution	Mean Peak Area
Standard 5.16 mg/L	2.8948 x 10^5
Standard 5.23 mg/L	2.9375 x 10^5
Initial Sample A, pH 4	2.3036 x 10^5
Initial Sample B, pH 4	2.4192 x 10^5
Initial Sample A, pH 7	2.3012 x 10^5
Initial Sample B, pH 7	2.4165 x 10^5
Initial Sample A, pH 9	2.4485 x 10^5
Initial Sample B, pH 9	2.5148 x 10^5
Standard 5.05 mg/L	2.7356 x 10^5
Standard 5.08 mg/L	2.7197 x 10^5
24 Hour Sample A, pH 4	1.4390 x 10^5
24 Hour Sample B, pH 4	1.5784 x 10^5
24 Hour Sample A, pH 7	1.5351 x 10^5
24 Hour Sample B, pH 7	1.5077 x 10^5
24 Hour Sample A, pH 9	1.4605 x 10^5
24 Hour Sample B, pH 9	1.5960 x 10^5
Standard 5.04 mg/L	2.5525 x 10^5
Standard 5.05 mg/L	2.5005 x 10^5
120 Hour Sample A, pH 4	5.0678 x 10^4
120 Hour Sample B, pH 4	3.6511 x 10^4
120 Hour Sample A, pH 7	4.6874 x 10^4
120 Hour Sample B, pH 7	4.5374 x 10^4
120 Hour Sample A, pH 9	3.8071 x 10^4
120 Hour Sample B, pH 9	4.7657 x 10^4

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

Table 7             pH 4 at 50 ºC - Repeat

Time (Hours)	Concentration (mg/L)		% of Mean Initial Concentration
	A	B	A	B
0	0.410	0.431	97.6	102
24	0.267	0.293	63.5	69.6
120	0.101	0.073	24.0	17.3

Table 8             pH 7 at 50 ºC - Repeat

Time (Hours)	Concentration (mg/L)		% of Mean Initial Concentration
	A	B	A	B
0	0.410	0.430	97.6	102
24	0.285	0.280	67.8	66.6
120	0.094	0.091	22.3	21.6

Table 9             pH 9 at 50 ºC - Repeat

Time (Hours)	Concentration (mg/L)		% of Mean Initial Concentration
	A	B	A	B
0	0.436	0.448	98.7	101
24	0.271	0.296	61.3	67.0
120	0.076	0.095	17.2	21.5

This test had a higher concentration of test item remaining at 24 and 120 hours.  This suggested that the higher percentage of organic solvent used as a co-solvent aided retention of the test item in solution.  As a significant amount of test item was still lost at each pH, it suggested that adsorption to the vessel caps was not a major contribution to reduction in concentration in solution.  It was considered that at 50 °C the test item was appreciably volatile, and this was the major contributor to loss of test item from solution.  Therefore, it was decided to perform a tier 2 test directly at 25 °C for a period of 28 days. 

Discussion

The preliminary/tier 1 test was initially performed using liquid chromatography mass spectrometry (LC-MS) for the sample analysis. The method had been shown to be acceptably linear from nominally 5 x 10^-3 to 0.1 mg/L for the appropriate matrices.  Timepoints were taken at 0, 24 and 120 hours also.  However, instrument response was variable at times, and it was considered not reliable enough for accurate quantitation.  It was probably that the method was sensitive to matrix variation even when solutions were matrix matched.  Additionally, a significant peak at the retention time of the test item was observed in all matrix blanks.  As a GC method had already been developed for the test item and considered reliable, it was decided to repeat the preliminary/tier 1 test using this analytical method.  As the GC method was significantly less sensitive than the LC-MS, a sample extraction procedure had to be developed and validated.  The results from the LC-MS analyses did indicate a loss in test item of similar magnitude as observed in the subsequent preliminary/tier 1 test.

The sample recovery procedure was performed for the sample preparation with 1% acetonitrile co-solvent only.  A separate procedure for the 5% acetone co-solvent was not performed.  However, it was considered that the procedure validated was adequately similar.  This is because some of the acetone would partition into the extracting hexane and the ionic nature of the sodium chloride would enhance partitioning of the test item and acetone into the hexane.  Also, as the test item has a high partition coefficient it would naturally favour the organic environment of the hexane solvent.

Various small peaks and baseline noise integration occurred in many of the GC chromatograms of the various matrix blanks.  The areas of these have not been reported.  As the peaks were often single entities and did not have a profile that matched that of the multi-component test item, they were considered to not represent the test item.  The matrix blanks were not subtracted from the standards or samples.

The repeated Preliminary/Tier 1 test used a higher percentage of co-solvent and that was less polar but still water soluble to investigate if this helped retain the test item in solution.  It had been considered that as the test item had a low water solubility, it could have been coming out of solution even though the test concentration was below the water solubility of the test item.  This would be more likely as the test solutions are prepared in buffers rather than purified water.  However, this process would have been expected to occur quickly rather than the gradual decrease over time.  The test temperature of 50 °C should also have enhanced solubility of the test item.

Alternatively, it was considered that the test item may have been volatizing from solution even though initially testing avoided a headspace above the sample in the test vessel.  Volatization would have been enhanced by the 50 °C test temperature and not unreasonable to occur gradually as this temperature is below the boiling point of the test item.  The 5% acetone co-solvent was also to try to minimize volatization, if lack of solubilization in the aqueous media was contributing to the issue.  Although the results for the repeated Preliminary/Tier 1 test still demonstrated a reduction in test item concentration for all three pHs, the loss was less than occurred using 1% acetonitrile co-solvent.

The decision for definitive testing was to perform the test directly at 25 °C.  This should then significantly reduce volatization and demonstrate what may happen in the environment even though the 5% acetone co-solvent was retained.  The test was conducted over a 28-day period to allow any changes to be distinctly observed and has been recommended by regulatory authorities previously.

One data point was removed from the correlation of the data points for both the pH 7 and pH 9 tests at 25 °C.  This was because they were considered inconsistent and to have a significant effect on the correlation.  They are plotted, colored red, on the graphical representation in Appendix 2 for reference.

The test item has no functional groups expected to be hydrolytically unstable at environmentally relevant temperatures and pH; therefore, the reduction in test item concentration in the samples was considered to be more likely due to   a physical loss rather than degradation.  Degradation results have been reported for information.

 

After 28 days, the estimated rate constant and half-life at 25 °C of the test item are shown in the following table:

Table 15

pH	Estimated Rate Constant at 25 °C (hr^-1)	Estimated Half-Life at 25 °C (days)	Most Test Item Lost from a Single Sample (%)
4	4.48 x 10^-4	64.5	36.6
7	3.42 x 10^-4	84.4	21.4
9	4.30 x 10^-4	67.2	28.8

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The hydrolytic stability of the test item was assessed. Although the test item had no functional groups expected to be hydrolytically unstable at environmentally relevant temperatures and pH, the concentration of test item in solution decreased over time; this was considered to be predominantly due to volatilization.

Executive summary:

Summary

The determination was carried out using a procedure designed to be compatible with Method C.7 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

Results/Conclusion

The hydrolytic stability of the test item was assessed.  Although the test item had no functional groups expected to be hydrolytically unstable at environmentally relevant temperatures and pH, the concentration of test item in solution decreased over time; this was considered to be predominantly due to volatilization.  After 28 days, the estimated rate constant and half-life at 25 °C of the test item are shown in the following table:

Table 15

pH	Estimated Rate Constant at 25 °C (hr^-1)	Estimated Half-Life at 25 °C (days)	Most Test Item Lost from a Single Sample (%)
4	4.48 x 10^-4	64.5	36.6
7	3.42 x 10^-4	84.4	21.4
9	4.30 x 10^-4	67.2	28.8