Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
In accordance with the preliminary test
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
In accordance with the preliminary test
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.2110 (Hydrolysis as a Function of pH)
Version / remarks:
In accordance with the preliminary test
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Melaleuca alternifolia, ext.
EC Number:
285-377-1
EC Name:
Melaleuca alternifolia, ext.
Cas Number:
85085-48-9
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Essential oil of melaleuca alternifolia
Test material form:
liquid
Specific details on test material used for the study:
- Physical state: Liquid.
- Purity: 100%
- Composition of test material: Composition meets ISO Standard 4730-2004 Oil of Melaleuca, terpinen-4-ol type (Tea Tree Oil). See below for detailed composition.
- Lot No.: 1215
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Details on sampling:
- Sampling method: The following procedure was carried out at each of pH 4, 7 and 9. The 100 ml sample of saturated aqueous solution of Tea Tree Oil was mixed with buffer solution (100 ml) to form a half-saturated solution. The resulting solution was then split into portions for the hydrolysis test so that 30 ml Wheaton vials were completely filled with the test solution. Separate vials were prepared for each timepoint in order to avoid loss of the test substance components due to volatility. One sample was analysed immediately and the remaining samples were placed in a 50°C water bath in the dark until sampling was required (after 2.4, 48 and 120 hours). At each sampling time, a single vial was removed from the water bath and duplicate portions (5 ml) were transferred to 10 ml volumetric flasks and diluted to volume with ethanol for analysis by gas chromatography (GC). Sample pH values and incubation temperature were monitored over the period of the test.
Buffers:
- pH: 4.0
- Type and final molarity of buffer: 0.04M with respect to phosphate
- Composition of buffer: Potassium dihydrogen orthophosphate (3.0 g) and disodium hydrogen orthophosphate dodecahydrate (6.4 g) were dissolved in purified water (950 ml) and the pH was adjusted to 4.0 ± 0.05 with orthophosphoric acid. The volume was then adjusted to 1000 ml with purified water.

- pH: 7.0
- Type and final molarity of buffer: 0.05M with respect to phosphate and 0.03M with respect to hydroxide
- Composition of buffer: Potassium dihydrogen orthophosphate (6.8 g) was dissolved in purified water (950 ml) and 1M sodium hydroxide (30 ml) was added to produce a solution of pH 7.0 ± 0.05. The volume was then adjusted to 1000 ml with purified water.

- pH: 9.0
- Type and final molarity of buffer: 0.044M with respect to borate and 0.013M with respect to phosphate
- Composition of buffer: Disodium tetraborate decahydrate (16.6 g) and potassium dihydrogen orthophosphate (1.8 g) were dissolved in purified water (950 ml) and the pH was adjusted to pH 9.0 ± 0.05. The volume was then adjusted to 1000 ml with purified water.
Details on test conditions:
- Preparation of aqueous stock solution of test substance: Since the test substance consisted of a mixture of components of varying solubility, a saturated aqueous solution of Tea Tree Oil was prepared. The procedure employed to prepare the stock solution was based on that used to prepare saturated solutions during water solubility testing (Huntingdon Life Sciences Report No. CSV0010/073174). A 2 litre aspirator was filled with purified water (2000 ml). A quantity (approximately 2.9 g) of the test substance was added to the surface of the water and the aspirator was sealed. The aspirator was then placed on a magnetic stirrer in a temperature controlled room at 20°C. Stirring was started and the speed was adjusted so that a minimal vortex was formed in order to avoid emulsion formulation. Triplicate samples (100 ml) were taken after stirring for approximately 24 hours. The stirrer was stopped for 1 hour prior to removal of samples of the saturated solution.
Duration of testopen allclose all
Duration:
120 h
pH:
4
Temp.:
50 °C
Duration:
120 h
pH:
7
Temp.:
50 °C
Duration:
120 h
pH:
9
Temp.:
50 °C
Number of replicates:
At each sampling time, a single vial was removed from the water bath and duplicate portions (5 ml) were transferred to 10 ml volumetric flasks and diluted to volume with ethanol for analysis by gas chromatography (GC).
Positive controls:
no
Negative controls:
no
Statistical methods:
The statistical methods of linear regressions and arithmetic means were used.

Results and discussion

Preliminary study:
There was no significant change in the concentration of Tea Tree Oil when incubated in pH 4, 7 and 9 buffer solutions at 50 ± 0.5°C (see Table 2 in 'Any other information on results'). Less than 10% hydrolysis had occurred after 120 hours (5 days) under these conditions, equivalent to a half-life of greater than 1 year under environmental conditions (25°C). Representative chromatograms from the test are presented in Figures 2 to 8 (see attached).
The pH values of test solutions over the period of the test are presented in Table 3 (see 'Any other information on results'). The results show that there were no significant changes in pH with time.
Transformation products:
not specified
Dissipation DT50 of parent compoundopen allclose all
Key result
pH:
4
Temp.:
50 °C
DT50:
> 1 yr
Type:
other:
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
7
Temp.:
50 °C
DT50:
> 1 yr
Type:
other:
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
9
Temp.:
50 °C
DT50:
> 1 yr
Type:
other:
Remarks on result:
hydrolytically stable based on preliminary test

Any other information on results incl. tables

Validation

Using the conditions described, the calibration of Tea Tree Oil was found to be linear over the concentration range 0 to 1140 mg/l, with a regression coefficient of 0.9995 (see Table 1 below, also attached Figure 1). No significant interfering peaks were evident in blank control solutions.

Table 1. Standard calibration for Tea Tree Oil.

 Standard concentration (mg/l)  Peak area
 45.54  7.3162
 91.09  17.606
 227.7  47.813
 455.4  100.65
 683.2  150.12
 910.9  204.99
 1139  265.66

Linear regression:              y = 0.232x - 3.62

(including x = 0, y = 0)       r = 0.9995

x = concentration

y = peak area

r = regression coefficient

Table 2. Preliminary investigation results for hydrolysis of Tea Tree Oil.

 pH  Ct (mg/l)
 t0h  t2.4h  t48h  t120h
 Measured Mean   Measured  Mean  Measured  Mean  Measured  Mean
 4  361.5, 359.7  360.6  353.7, 354.3  354.0  348.6, 345.4  347.0  372.9, 372.1  372.5
 7  355.9, 366.8  361.4  361.0, 547.7*  361.0  349.7, 351.5  350.6  393.9, 379.6  386.8
 9  352.8, 370.9  361.8  361.0, 361.6  361.3  361.6, 360.6  361.1  390.3, 385.8  388.0

Where Ct is the concentration of Tea Tree Oil in solution at time th (in hours).

* value not included in mean since it was anomalously high.

Table 3. pH during preliminary hydrolysis tests.

 Nominal pH  Initial pH  Final pH
 4  4.4  4.3
 7  7.2  7.1
 9  9.0  9.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Tea Tree Oil was determined to be hydrolytically stable under acidic, neutral and basic conditions. On examination of the structures of the major components of the test substance, it was evident that they did not contain hydrolysable functional groups, thereby supporting the experimental findings of the study.
Executive summary:

A study was performed to determine the rate of hydrolysis of Tea Tree Oil as function of pH. The study was performed in accordance with the preliminary test as described in the EEC Method C7, OECD Method 111, and EPA/OPPTS 835.2110. The preliminary study showed that at pH 4, 7 and 9 and 50 ± 0.5°C, less than 10% hydrolysis had occurred after 120 hours (5 days), equivalent to a half-life of greater than 1 year under environmental conditions (25°C). Tea Tree Oil was determined to be hydrolytically stable under acidic, neutral and basic conditions.