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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 4, 2003 to May 28, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
temperature, one replicate
GLP compliance:
yes
Specific details on test material used for the study:
Parent compound 3-Chloro-o-xylene was abbreviated with 3CX.
Transformation product 2,3-dimethylphenol was abbreviated with DMP.
Co-solvent acetonitrile was abbreviated with ACN or CH3CN.
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products: only once after 5 days.
- Sampling intervals/times for pH measurements: at preparation
Buffers:
pH 4 Buffer: An acetate buffer was prepared by dissolving 1.155 mL of glacial acetic acid in 1 L of deionized water (DIW) (solution A; 0.02 M). Solution B was prepared by dissolving 1.64 g of anhydrous sodium acetate (or 2.72 g of the trihydrate) in 1.000 L of DIW (0.02 M). Solution A (41.0 mL) and Solution B (9.0 mL) were mixed. The pH of this buffer was measured with a pH meter. This buffer (26.5 mL) was mixed with 100 mL of DIW (5 mM buffer). The pH of the diluted buffer was 3.55.

pH 7 Buffer: MOPS (3-N-morpholino propanesulfonic acid) free acid (20.93 g; 0.05 M) was dissolved in 2 L of DIW. The solution was titrated to pH 7.0 with approx. 20 mL of 1.00 N aqueous NaOH. This buffer (10.0 mL) was mixed with 100 mL of DIW (5 mM buffer). The pH of the diluted buffer was 6.51.

pH 9 Buffer: Dissolved 1.222 g of ethanolamine (0.02 M) in approx. 900 ml of deionized water. The solution was titrated to pH 9.0 with approx. 32 mL of ca. 1.0 M HCI, and made up to 1000 mL with deionized water. This buffer (26.5 mL) was mixed with 100 mL of DIW (5 mM buffer). The pH of the diluted buffer was 8.74.

Sk3CX9: test item solution in CH3CN: 3CX (28.87 mg) was dissolved in 25.00 mL of CH3CN in a volumetric flask (1.1548 mg/mL).

SkDM1: 2,3-Dimethylphenol (DMP) solution in CH3CN: DMP (10.11 mg) was dissolved in 10.00 mL of CH3CN in a volumetric flask (1.0110 mg/mL).
Details on test conditions:
TEST MEDIUM
- Kind and purity of water: Millipore Elix System, Milford, MA
- Preparation of test medium: see in IUCLID field 'Buffer' or below.
- Renewal of test solution: no, terminated after 5 days
- Identity and concentration of co-solvent: Acetonitrile (CH3CN), 0.5 % in final hydrolysis test solution

TEST SOLUTIONS
The K solutions, G 'Hydrolysis Solutions' and J 'Control Solutions' were prepared as as indicated in the table provided in 'any other information on materials and methods'. The G solutions were placed at 55 ± 1 °C for 5.0 d, mixing horizontally at 1 rps. The B and R solutions were prepared as as indicated in the table in 'any other information on materials and methods' and stored (for 5 d) at ambient temperature awaiting HPLC.

OTHER TEST CONDITIONS
- Adjustment of pH: no

Additional Test: Adsorption Experiment
An additional experiment was conducted to investigate the adsorption of the test substance onto glass. These results were required to support the hydrolysis test results. For details see any other information on materials and methods.
Duration:
5 d
pH:
4
Temp.:
55 °C
Initial conc. measured:
0.72 mg/L
Duration:
5 d
pH:
7
Temp.:
55 °C
Initial conc. measured:
0.84 mg/L
Duration:
5 d
pH:
9
Temp.:
55 °C
Remarks:
below detection limit (DL)
Number of replicates:
N=1 per pH and per temperature
Positive controls:
no
Negative controls:
no
Statistical methods:
Statistical calculations (standard deviation, average, histograms, correlation coefficients) were performed using Microsoft Excel software.
Preliminary study:
A preliminary hydrolysis test was performed as described in the OECD TG 111 (see also test performance in the field below).
Test performance:
For the test item hydrolysis solutions, the temperatures of ambient air and the (55 °C) incubator were recorded every 9 min using thermocouples immersed in ca. 2 mL of oil ('G solutions'). The thermocouples were calibrated by measuring the temperatures of the thermocouples 25.8 ± 0.0 °C (n = 3) and a calibrated thermometer 25.9 ± 0.0 °C (n = 3) equilibrated to ambient temperature. The thermocouple deviation of 0.1 °C was ignored.
Concurrently, three other test item hydrolysis solutions (one at pH 4, 7 and 9) were frozen horizontally for 5 d ('J solutions'). After 5 d, the J solutions were thawed and vortex mixed for ca. 10 s before HPLC.
Transformation products:
no
Details on hydrolysis and appearance of transformation product(s):
For details on results for hydrolysis and transformation product of the test substance as well as the adsorption experiment see any other information on results.
% Recovery:
13
pH:
4
Temp.:
55 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
% Recovery:
15
pH:
7
Temp.:
55 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
% Recovery:
0
pH:
9
Temp.:
55 °C
Duration:
5 d
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
4
Temp.:
55 °C
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
7
Temp.:
55 °C
Remarks on result:
hydrolytically stable based on preliminary test
Key result
pH:
9
Temp.:
55 °C
Remarks on result:
hydrolytically stable based on preliminary test
Details on results:
The HPLC results indicated that the observed test item concentrations in the heated and frozen solutions were lower than nominal. However, the lower test item concentrations were not due to hydrolysis of 3-chloro-o-xylene, because no hydrolysis product (DMP) was detected.
These lower-than-nominal 3-chloro-o-xylene concentrations were likely due to the adsorption of the test item to the vial glass, which was supported by results from an additional experiment that examined the effect of varying the co-solvent (CH3CN) concentration on the observed test item concentration in pH 9 buffer solutions (see any other information on materials and methods). In this experiment, the observed 3-chloro-o-xylene concentration generally decreased as the CH3CN concentration decreased. It was concluded that 3-chloro-o-xylene was hydrolytically stable as defined by the OECD 111 (half-life > 1 year at 25 °C).

Observed test item concentrations in hydrolysis solutions.

Label

HPLC Peak
Area

Test item Conc.
(ug/mL)

% of Initial Nominal
Test Item
Conc.

RA

698.0

5.82

101%

R4

258.5

2.18

38%

R7

264.1

2.23

39%

R9

243.4

2.06

36%

J4

233.1

1.97

34%

J7

249.1

2.10

37%

J9

206.5

1.75

30%

G4

82.2

0.72

13%

G7

96.4

0.84

15%

G9

0.0

< DL

0%

"R" solutions: Spiked with test item and DMP (2,3-dimethylphenol) (to demonstrate chromatography).

"G" solutions: Hydrolysis solutions, heated at 55 °C.

"J" solutions: Control solutions, concurrently frozen.

Adsorption Experiment: Observed relative test item concentrations in adsorption solutions.

Label Test item peak area Duplicate area Normalized area
(%)

Acetonitrile conc.
(vol. %)

AAa 0 0% 0.5%
AAb 343.275 49%

0.5%

BAa 271.057 38% 2.0%
BAb 255.872 36% 2.0%
CAa 125.172 18% 10.0%
CAb 112.148 16% 10.0%
DAa 711.995 101% 50.0%
DAb 709.3721 101% 50.0%
EAa 706.353 100% 100.0%
EAb 703.375 704.864 100% 100.0%
AHa 294.159 40% 0.5%
AHb 236.954 32% 0.5%
BHa 221.977 30% 2.0%
BHb 199.846 27% 2.0%
CHa 330.583 45% 10.0%
CHb 0 0% 10.0%
DHa 704.9003 95% 50.0%
DHb 708.6846 95% 50.0%
EHa 731.756 99% 100.0%
EHb 753.773 742.764 101% 100.0%
Validity criteria fulfilled:
yes
Remarks:
(as far as applicable for hydrolytically stable compounds)
Conclusions:
3-Chloro-o-xylene is hydrolytically stable.
Executive summary:

The OECD TG 111, hydrolysis screening test was conducted in compliance with GLP, in which test item solutions in buffers (pH 4, 7, and 9) containing 0.5 % acetonitrile co-solvent were heated at 55 °C for 5 days, and an identical set of solutions was concurrently frozen. The HPLC results indicated that the observed test item concentrations in the heated and frozen solutions were lower than nominal. However, this was not due to the hydrolysis of 3-chloro-o-xylene, because no hydrolysis product, 2,3-dimethylphenol (DMP), was detected. These lower-than-nominal test item concentrations were likely due to the adsorption of 3-chloro-o-xylene to the vial glass, which was supported by results from an additional adsorption experiment. The adsorption experiment examined the effect of varying co-solvent (CH3CN) concentrations on the observed test item concentration in pH 9 buffer solutions. In fact, the observed test item concentration generally decreased as the acetonitrile concentration decreased.

Therefore, it was concluded that based on the results of this study, 3-chloro-o-xylene was hydrolytically stable as defined by the OECD TG 111 (half-life > 1 year at 25 °C).

Despite of deviation from the test guideline (single replicates, 55°C instead of 50°C test temperature), the result of the study were considered reliable and adequate for the environmental hazard assessment.

Description of key information

3-Chloro-o-xylene is hydrolytically stable (OECD 111, 2004).

Key value for chemical safety assessment

Additional information

A reliable key study was conducted according to OECD TG 111, hydrolysis screening test (RL1, 2004). The study was conducted in compliance with GLP. The HPLC results indicated that the observed test item concentrations in heated and frozen test solutions were lower than nominal. However, this was not due to the hydrolysis of 3-chloro-o-xylene, because no hydrolysis product was detected. These lower-than-nominal test item concentrations were likely due to the adsorption of 3-chloro-o-xylene to the vial glass, which was supported by results from an additional adsorption experiment. The adsorption experiment examined the effect of varying co-solvent concentrations on the observed test item concentration. In fact, the observed test item concentration generally decreased as the acetonitrile concentration decreased.

Therefore, it was concluded that based on the results of the study, 3-chloro-o-xylene was hydrolytically stable (half-life > 1 year at 25 °C). Despite of minor deviation from the test guideline, results of the study were considered reliable and adequate for the environmental hazard assessment.