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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - October 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Not according to OECD111, no GLP, however CoA available and sufficient reporting on methods and results
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
Tier 1 and Tier 3 at adjusted temperature (37C), pH and duration (6h), no GLP, citric acid phosphate and carbonate buffer solutions. Including pH 2.2
GLP compliance:
yes
Specific details on test material used for the study:
Test item DMOE Ac
3,7-dimethyl-1-octen-3-yl acetate
DSM number DSM078139
CAS number 68345-17-5
Synonyms 3,7-dimethyloct-1-en-3-yl acetate
Formula C12H22O2
Molecular weight 198.31 g/mol
Boiling point 238 °C (calculated, ACD/Labs V12.01)
Water solubility 0.17 g/l, 0.86 mmol/l (calculated, ACD/Labs V12.01)
Log P 4.25 (calculated, ACD/Labs V12.01)

Radiolabelled test item 14C-DMOE Ac
[3-14C]DSM078139
(R,S)-[3-14C]-3,7-dimethyl-1-octen-3-yl acetate
Batch/Lot-No. 7688SJR003-2
Supplier Selcia Limited, Ongar, Essex, UK
Specific activity 31.85 mCi/mmol (1178 MBq/mmol)
159.8 uCi/mg (5.91 MBq/mg)
Storage Supplied as a solution in ethanol with a concentration of:
1.074 mCi/ml (39.75 MBq/ml)
Radiochemical purity 99.4% (HPLC, November 2013)
Chemical purity Contains 0.1% solvents (NMR, November 2013)
Storage conditions Glass bottle, <-15°C
Radiolabelling:
yes
Analytical monitoring:
yes
Details on sampling:
Series 1
50 ul of the DMOE Ac stock solution was added to 450 ul buffer and immediately 50 ul aliquots
were distributed into 8 individual conical HPLC vials (450 μL glass insert, fused into a 2 ml
crimp top vial from Chromacol, UK). The HPLC vials were crimp capped with a PTFE seal and
placed into the HPLC autosampler thermostatted at 37°C. At each sampling time point 20 ul
aliquots were removed (one aliquot from a fresh vial) and analyzed by HPLC (S1).
The concentration of DMOE Ac in the incubation solution corresponded to 0.13 mmol/l or 9540
dpm/ul.

Series 2 &3
50 ul of the DMOE Ac or DMOE stock solution was added to 450 ul buffer and immediately
about 400 - 450 ul was filled into one conical HPLC vial (450 μL glass insert, fused into a 2 ml
crimp top vial from Chromacol, UK) leaving as less void volume as possible. The HPLC vials
were crimp capped with a PTFE seal and placed into the HPLC autosampler thermostatted at
37°C. At each sampling time point 20 ul aliquots were removed from the same vial and analyzed
by HPLC (S1 for Series 2 and S2 for Series 3).
The concentration of DMOE Ac in the incubation solution corresponded to 0.13 mmol/l or 9540
dpm/ul. For DMOE the concentration was 0.13 mmol/l or 8866 dpm/ul.

Series 4
40 ul aliquots of the DMOE Ac stock solution were added into 3 20 ml headspace vials
containing 960 ul buffer and were crimp capped with a PTFE seal and placed into the incubator
of the headspace autosampler. The incubation was performed at 37°C. At each sampling time
point the headspace vials were removed and analyzed by Headspace M.
Immediately after the measurement the remaining samples were frozen and stored at -20°C.
For analysis of the aqueous phase 20 ul aliquots were removed and analyzed by HPLC (S1).
The concentration of DMOE Ac in the incubation solution corresponded to 0.13 mmol/l or 9540
dpm/ul.
Buffers:
pH 2.2: 98.8% 0.1 mol/l citric acid monohydrate + 1.2% 0.2 mol/l disodium hydrogen phosphate
pH 4.5: 56.4% 0.1 mol/l citric acid monohydrate + 43.6% 0.2 mol/l disodium hydrogen phosphate
pH 7.5: 19.0% 0.1 mol/l citric acid monohydrate + 81% 0.2 mol/l disodium hydrogen phosphate
pH 9.2: 10.0% 0.1 mol/l sodium carbonate + 90.0% 0.1 mol/l sodium hydrogen carbonate
Duration:
6 h
pH:
9.2
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 1
Duration:
3 h
pH:
2.2
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 1
Duration:
6 h
pH:
9.2
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 2
Duration:
6 h
pH:
7.5
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 2
Duration:
6 h
pH:
4
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 2
Duration:
6 h
pH:
2.2
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 2
Duration:
2 h
pH:
2.2
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 3
Duration:
6 h
pH:
9.2
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 4
Duration:
6 h
pH:
4
Temp.:
37 °C
Initial conc. measured:
0.13 mmol/L
Remarks:
series 4
Number of replicates:
single samples
Positive controls:
no
Negative controls:
no
Transformation products:
yes
Remarks:
Only DMOE was identified
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
 DMOE Ac was non-enzymatically hydrolyzed to DMOE at the temperature and pH range corresponding to physiological conditions.
 Hydrolysis was faster at low pH than at high pH.
 No other reaction products were detected.
% Recovery:
32
pH:
9.2
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 1
% Recovery:
69
pH:
2.2
Temp.:
37 °C
Duration:
3 h
Remarks on result:
other: loss due to volatization
Remarks:
series 1
% Recovery:
48
pH:
9.2
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
66
pH:
7.5
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
52
pH:
4
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
80
pH:
2.2
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
77
pH:
2.2
Temp.:
37 °C
Duration:
2 h
Remarks on result:
other: loss due to volatization
Remarks:
series 3
pH:
9.2
Temp.:
37 °C
Hydrolysis rate constant:
0.239 h-1
DT50:
2.9 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
Key result
pH:
7.5
Temp.:
37 °C
Hydrolysis rate constant:
0.203 h-1
DT50:
3.41 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
pH:
4
Temp.:
37 °C
Hydrolysis rate constant:
0.292 h-1
DT50:
2.37 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
pH:
2.2
Temp.:
37 °C
Hydrolysis rate constant:
1.95 h-1
DT50:
0.36 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
pH:
2.2
Temp.:
37 °C
Hydrolysis rate constant:
2.553 h-1
DT50:
0.27 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 3
Details on results:
The incubation of 14C-DMOE Ac with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the
following results:
 DMOE Ac was non-enzymatically hydrolyzed to DMOE at the temperature and pH range
corresponding to physiological conditions.
 Hydrolysis was faster at low pH than at high pH. This indicates that in the stomach
DMOE Ac will be rapidly and completely hydrolyzed to DMOE due to the low pH
conditions.
 No other reaction products were detected.
 At 37°C a significant loss of DMOE Ac due to volatilization was observed.
The incubation of 14C-DMOE with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the
following results:
 DMOE is chemically stable under the conditions tested.
 DMOE is only slightly volatile in aqueous systems.
Validity criteria fulfilled:
not specified
Conclusions:
The incubation of 14C-DMOE Ac with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the following results:
 DMOE Ac was non-enzymatically hydrolyzed to DMOE at the temperature and pH range corresponding to physiological conditions.
 Hydrolysis was faster at low pH than at high pH, with half lives of 2.9, 3.4, 2.4 and 0.3 h at pH 9.2, 7.5, 4.0 and 2.2 respectively
 No other reaction products were detected.
 At 37°C a significant loss of DMOE Ac due to volatilization was observed.
The incubation of 14C-DMOE with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the following results:
 DMOE is chemically stable under the conditions tested.
 DMOE is only slightly volatile in aqueous systems.
Executive summary:

Data for hydrolysis for ethyllinalyl acetate were obtained from the read across substance 3,7-dimethyl-1-octen-3-yl acetate (DMOE Ac).

The chemical stability of 3,7-dimethyl-1-octen-3-yl acetate (DMOE Ac) was tested in aqueous solutions under different physiological conditions, i.e. at different pH values in the range of pH 2.2-9.2 at 37°C to mimic the hydrolysis behavior in the different parts of the gastrointestinal tract.

[3-14C]-3,7-dimethyl-1-octen-3-yl acetate (14C-DMOE Ac) was incubated at a concentration of 0.13 mmol/L at 37°C in buffers at pH 9.2, 7.5, 4.0 and 2.2. At several time points aliquots were removed and analyzed by radio-HPLC.

In the aqueous phase DMOE Ac was hydrolyzed to DMOE (3,7-dimethyl-1-octen-3-ol) with half lives of 2.9, 3.4, 2.4 and 0.3 h at pH 9.2, 7.5, 4.0 and 2.2 respectively. Thus, below pH 7.5 the hydrolysis rate was inversely related to the pH: the lower the pH, the higher the rate. Besides the hydrolysis product DMOE, no other degradation products were detected. Although closed systems were used and the gas volume in the incubation vials was kept low, the recovery of radioactivity in the aqueous phase decreased to about 50% after 6h, indicating a loss of volatile compounds at higher pH values. However, at pH 2.2 the overall radioactivty in the aqueous phase decreased only to 80% of the starting amount within 6h.

In control experiments, 14C-DMOE was incubated under identical conditions. DMOE was stable for up to 6h. The recovery of radioactivity was > 83% indicating that in aqueous solutions DMOE Ac was more volatile than DMOE. This was confirmed by measuring the ratio of DMOE Ac to DMOE in the gas phase by headspace MS. A ratio of about 90/10 was found in the gas phase after 6h incubation.

The results show that non-enzymatic hydrolysis of DMOE Ac to DMOE is faster at the lowest pH than at the highest pH tested. This indicates that in the stomach DMOE Ac will be rapidly (the calculated half-life is 0.3h) and completely hydrolyzed to DMOE due to the low pH conditions.

Endpoint:
hydrolysis
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Transformation products:
yes
Remarks:
Only DMOE was identified
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
 DMOE Ac was non-enzymatically hydrolyzed to DMOE at the temperature and pH range corresponding to physiological conditions.
 Hydrolysis was faster at low pH than at high pH.
 No other reaction products were detected.
% Recovery:
32
pH:
9.2
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 1
% Recovery:
69
pH:
2.2
Temp.:
37 °C
Duration:
3 h
Remarks on result:
other: loss due to volatization
Remarks:
series 1
% Recovery:
48
pH:
9.2
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
66
pH:
7.5
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
52
pH:
4
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
80
pH:
2.2
Temp.:
37 °C
Duration:
6 h
Remarks on result:
other: loss due to volatization
Remarks:
series 2
% Recovery:
77
pH:
2.2
Temp.:
37 °C
Duration:
2 h
Remarks on result:
other: loss due to volatization
Remarks:
series 3
pH:
9.2
Temp.:
37 °C
Hydrolysis rate constant:
0.239 h-1
DT50:
2.9 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
Key result
pH:
7.5
Temp.:
37 °C
Hydrolysis rate constant:
0.203 h-1
DT50:
3.41 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
pH:
4
Temp.:
37 °C
Hydrolysis rate constant:
0.292 h-1
DT50:
2.37 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
pH:
2.2
Temp.:
37 °C
Hydrolysis rate constant:
1.95 h-1
DT50:
0.36 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 2
pH:
2.2
Temp.:
37 °C
Hydrolysis rate constant:
2.553 h-1
DT50:
0.27 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: series 3
Details on results:
The incubation of 14C-DMOE Ac with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the
following results:
 DMOE Ac was non-enzymatically hydrolyzed to DMOE at the temperature and pH range
corresponding to physiological conditions.
 Hydrolysis was faster at low pH than at high pH. This indicates that in the stomach
DMOE Ac will be rapidly and completely hydrolyzed to DMOE due to the low pH
conditions.
 No other reaction products were detected.
 At 37°C a significant loss of DMOE Ac due to volatilization was observed.
The incubation of 14C-DMOE with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the
following results:
 DMOE is chemically stable under the conditions tested.
 DMOE is only slightly volatile in aqueous systems.
Validity criteria fulfilled:
not specified
Conclusions:
The incubation of 14C-DMOE Ac with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the following results:
 DMOE Ac was non-enzymatically hydrolyzed to DMOE at the temperature and pH range corresponding to physiological conditions.
 Hydrolysis was faster at low pH than at high pH, with half lives of 2.9, 3.4, 2.4 and 0.3 h at pH 9.2, 7.5, 4.0 and 2.2 respectively
 No other reaction products were detected.
 At 37°C a significant loss of DMOE Ac due to volatilization was observed.
The incubation of 14C-DMOE with buffers at pH 2.2, 4.0, 7.5 and 9.2 at 37°C showed the following results:
 DMOE is chemically stable under the conditions tested.
 DMOE is only slightly volatile in aqueous systems.
Executive summary:

Data for hydrolysis for ethyllinalyl acetate were obtained from the read across substance 3,7-dimethyl-1-octen-3-yl acetate (DMOE Ac).

The chemical stability of 3,7-dimethyl-1-octen-3-yl acetate (DMOE Ac) was tested in aqueous solutions under different physiological conditions, i.e. at different pH values in the range of pH 2.2-9.2 at 37°C to mimic the hydrolysis behavior in the different parts of the gastrointestinal tract.

[3-14C]-3,7-dimethyl-1-octen-3-yl acetate (14C-DMOE Ac) was incubated at a concentration of 0.13 mmol/L at 37°C in buffers at pH 9.2, 7.5, 4.0 and 2.2. At several time points aliquots were removed and analyzed by radio-HPLC.

In the aqueous phase DMOE Ac was hydrolyzed to DMOE (3,7-dimethyl-1-octen-3-ol) with half lives of 2.9, 3.4, 2.4 and 0.3 h at pH 9.2, 7.5, 4.0 and 2.2 respectively. Thus, below pH 7.5 the hydrolysis rate was inversely related to the pH: the lower the pH, the higher the rate. Besides the hydrolysis product DMOE, no other degradation products were detected. Although closed systems were used and the gas volume in the incubation vials was kept low, the recovery of radioactivity in the aqueous phase decreased to about 50% after 6h, indicating a loss of volatile compounds at higher pH values. However, at pH 2.2 the overall radioactivty in the aqueous phase decreased only to 80% of the starting amount within 6h.

In control experiments, 14C-DMOE was incubated under identical conditions. DMOE was stable for up to 6h. The recovery of radioactivity was > 83% indicating that in aqueous solutions DMOE Ac was more volatile than DMOE. This was confirmed by measuring the ratio of DMOE Ac to DMOE in the gas phase by headspace MS. A ratio of about 90/10 was found in the gas phase after 6h incubation.

The results show that non-enzymatic hydrolysis of DMOE Ac to DMOE is faster at the lowest pH than at the highest pH tested. This indicates that in the stomach DMOE Ac will be rapidly (the calculated half-life is 0.3h) and completely hydrolyzed to DMOE due to the low pH conditions.

Description of key information

Data for hydrolysis for ethyllinalyl acetate were obtained from the read across substance 3,7-dimethyl-1-octen-3-yl acetate (DMOE Ac).

The chemical stability of 3,7-dimethyl-1-octen-3-yl acetate (DMOE Ac) was tested in aqueous solutions under different physiological conditions, i.e. at different pH values in the range of pH 2.2-9.2 at 37°C to mimic the hydrolysis behavior in the different parts of the gastrointestinal tract.

[3-14C]-3,7-dimethyl-1-octen-3-yl acetate (14C-DMOE Ac) was incubated at a concentration of 0.13 mmol/L at 37°C in buffers at pH 9.2, 7.5, 4.0 and 2.2. At several time points aliquots were removed and analyzed by radio-HPLC.

In the aqueous phase DMOE Ac was hydrolyzed to DMOE (3,7-dimethyl-1-octen-3-ol) with half lives of 2.9, 3.4, 2.4 and 0.3 h at pH 9.2, 7.5, 4.0 and 2.2 respectively. Thus, below pH 7.5 the hydrolysis rate was inversely related to the pH: the lower the pH, the higher the rate. Besides the hydrolysis product DMOE, no other degradation products were detected. Although closed systems were used and the gas volume in the incubation vials was kept low, the recovery of radioactivity in the aqueous phase decreased to about 50% after 6h, indicating a loss of volatile compounds at higher pH values. However, at pH 2.2 the overall radioactivty in the aqueous phase decreased only to 80% of the starting amount within 6h.

Key value for chemical safety assessment

Half-life for hydrolysis:
3.4 h
at the temperature of:
37 °C

Additional information

The hydrolysis half-life of 3.4 hours was concluded from data at pH 7.5 which is considered representative for environmental aquatic conditions.

ELAC and DMOEAc are well-defined, fall in the allylic esters class and show high similarity in structure and properties. Therefore, the hydrolysis can be predicted because a similar reaction for these two substances is anticipated. The predicted values for hydrolysis half-lives are between 0.3 and 3.4 hours and can be used for ELAC. Experimental data from the OECD SIDS program for another close analogue, LAC, includes results in the same order of magnitude which further validate the hypothesis.