Registration Dossier

Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 February 2011 – 30 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and EC guidelines and in compliance with GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
(2004)
Deviations:
yes
Remarks:
Temperature criterion of +/- 0.5°C was not met during main test pH 9 at 20°C. For some 2.5 h an increase to max 21.9°C occurred. As this period was <1% of the duration of the pH9 main test, the temp rise was concluded to have no effect on the est.d t ½.
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
(Official Journal of the European Union no. L142, May 31, 2008)
Deviations:
yes
Remarks:
; see OECD 111
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
E-BK105
IUPAC Name:
E-BK105
Details on test material:
- Name of test material (as cited in study report): E-BK105
- Description: Black powder
- Batch: MB-1
- Test substance storage: At room temperature protected from light
- Stability under storage conditions: Stable
- Expiry date: 23 December 2015
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Details on sampling:
The concentration of the test substance in the test samples was determined immediately after preparation (t=0) and after 5 days (preliminary test) or 13 days at the most (main test pH 9). The samples taken at t=5 days were cooled to room temperature using running tap water. The samples were diluted in a 1:1 (v:v) ratio with 10 mM TBABr in acetonitrile and thereafter further diluted by a factor of 50 with 5 mM TBABr in 50/50 (v/v) acetonitrile/water and analysed.

Blank buffer solutions were treated similarly as the test samples and analysed at t=0.

The pH of each of the test solutions (except for the blanks) was determined at each sampling time.
Buffers:
- pH: 4
- Type and final molarity of buffer: Acetate buffer, 0.1 M
- Composition of buffer: solution of 16.6% 0.1 M sodium acetate and 83.4% 0.1 M acetic acid. The buffer contains 0.0009% (w/v) sodium azide.

- pH: 7
- Type and final molarity of buffer: Phosphate buffer, 0.1 M
- Composition of buffer: solution of 0.1 M potassium dihydrogenphosphate adjusted to pH 7 using 10 N sodium hydroxide. The buffer contains
0.0009% (w/v) sodium azide.

- pH: 9
- Type and final molarity of buffer: Borate buffer, 0.1 M
- Composition of buffer: solution of 0.1 M boric acid and 0.1 M potassium chloride adjusted to pH 9 using 10 N sodium hydroxide. The buffer
contains 0.0009% (w/v) sodium azide.
Details on test conditions:
Preliminary test - Tier 1:

Test substance solutions were prepared in the buffer solutions at a target concentration of 100 mg/l. Each solution was filter-sterilised through a 0.2 μm FP 30/0.2 CA-S filter (Whatman, Dassel, Germany) and transferred into a sterile vessel. To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 ml test solution and placed in the dark in a temperature controlled environment at 49.9°C +/- 0.1°C.

Main study - Tier 2:

Test samples were prepared in the buffer solution pH 9 at a target concentration of 1000 mg/l and treated similarly as during the preliminary test. Compared to the preliminary test, E-BK105 concentration was increased by a factor 10 for detection of concentrations down to 10% of the start
concentration i.e. up to 90% hydrolysis.

The main study (pH9) was performed with the following temperatures:
temp I: 19.9°C +/- 0.5°C
temp II: 49.9°C +/- 0.2°C
temp III: 70.1°C +/- 0.2°C

The temperature during testing at 20°C was between 19.6°C and 20.4°C except for a 2.5-hours period in which the temperature increased to maximum 21.9°C. This time period was < 1% of the duration of the the main test pH 9 at 20°C. The temperature rise had no effect on the estimated half-life time and was therefore accepted. This deviation did not adversely impact the overall outcome of this study.
Duration of testopen allclose all
Duration:
5 d
pH:
4
Initial conc. measured:
74.3 - 76.2 mg/L
Duration:
5 d
pH:
7
Initial conc. measured:
109 - 113 mg/L
Duration:
5 d
pH:
9
Initial conc. measured:
101 - 102 mg/L
Duration:
305 h
pH:
9
Initial conc. measured:
1 028 - 1 043 mg/L
Number of replicates:
2 measurements at each pH (separate vessels)

Results and discussion

Preliminary study:
At pH 4 and pH 7, a degree of hydrolysis of < 10% was observed after 5 days. It demonstrated that the half-life time of the test substance at 25°C is > 1 year. According to the guideline, no further tests were required.

At pH 9, a degree of hydrolysis of > 10% after 5 days was observed (i.e. 15%). According to the guideline, the higher Tier 2 test was required to determine the half-life time of the test substance.

A small response at the retention time of the test substance was observed in the blank buffer solutions. This was also observed in water and 5 mM TBABr in 50/50 (v/v) acetonitrile/water solutions (see also section 8, HPLC-UV: specificity). Therefore, it was considered that the response did not derive from the test substance.
Test performance:
The mean recoveries of the buffer solutions (at time 0, preliminary test) fell within the acceptable range for non labelled chemicals of 70-110%: pH4 - 75%, pH7 - 107%, pH9 - 101%. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test substance.
Total recovery of test substance (in %)open allclose all
% Recovery:
74 - 76
pH:
4
Temp.:
49.9 °C
% Recovery:
105 - 109
pH:
7
Temp.:
49.9 °C
% Recovery:
100 - 101
pH:
9
Temp.:
49.9 °C
Details on results:
Main study (Tier 2) at 20, 50 and 70°C:

No test substance was detected in the blank buffer solutions.

The results of the Tier 2 test indicated that no significant hydrolysis (< 10%) is observed at 20 and 50°C over a period of 13 days (305 hours). At 70°C, a mean of 40% hydrolysis was observed after 13 days (305 hours).

In the main test (up to 305 hours) at 50°C, the degree of hydrolysis was observed to be < 10% which suggests that the half-life of the test substance at 25°C at pH 9 would be > 1 year. For this reason the Tier 2 test was stopped at day 13 (305 hours) and not continued for 30 days or until
90% hydrolysis was observed.

The recoveries of the test solution used in the tests at all three test temperatures fell within the criterion range of 90-110%.

Any other information on results incl. tables

Preliminary test – hydrolysis of the test substance at pH 4, pH 7 and pH 9

pH code

Sampling time

Analysed concentration
[mg/l]

Degree of hydrolysis
[%]

Actual pH

Individual

Mean

 

 

 

 

 

 

pH 4

0 hours

76.2

 

 

4.0

 

 

74.3

 

 

4.0

 

5 days

79.8

-6.1

-3.5

4.0

 

 

75.9

-0.8

 

4.0

 

 

 

 

 

 

pH 7

0 hours

113

 

 

7.1

 

 

109

 

 

7.1

 

5 days

111

-0.2

0.5

7.1

 

 

110

1.3

 

7.1

 

 

 

 

 

 

pH 9

0 hours

101

 

 

9.0

 

 

102

 

 

9.0

 

5 days

87.1

14

15

9.0

 

 

85.6

16

 

9.0

 

 

 

 

 

 

Main test – hydrolysis of the test substance at 20°C

Sampling time

[hours]

 

Analysed

concentration

[mg/]

Relative

concentration

[%]

Logarithm relative

concentration

 

Actual pH

 

0

1028

99

2.00

9.0

0

1043

101

2.00

9.1

65

1093

105

2.02

9.1

65

1097

106

2.03

9.1

185

1037

100

2.00

9.0

185

1044

101

2.00

9.0

305

1076

104

2.02

9.0

305

1074

104

2.02

9.0

Main testhydrolysis of the test substance at 50°C

Sampling time

[hours]

 

Analysed

concentration

[mg/]

Relative

concentration

[%]

Logarithm relative

concentration

 

Actual pH

 

0

1028

99

2.00

9.0

0

1043

101

2.00

9.1

65

1034

100

2.00

9.1

65

1033

100

2.00

9.1

185

964

93

1.97

9.0

185

962

93

1.97

9.0

305

971

94

1.97

9.0

305

959

93

1.97

9.0

Main testhydrolysis of the test substance at 70°C

Sampling time

[hours]

 

Analysed

concentration

[mg/]

Relative

concentration

[%]

Logarithm relative

concentration

 

Actual pH

 

0

1028

99

2.00

9.0

0

1043

101

2.00

9.1

17

942

91

1.96

9.1

17

883

85

1.93

9.1

25

891

86

1.93

9.1

25

879

85

1.93

9.1

41

869

84

1.92

9.1

41

974

84

1.93

9.1

65

1093

83

1.92

9.1

65

1097

82

1.92

9.1

185

1037

68

1.83

9.0

185

1044

67

1.82

9.0

305

1076

51

1.71

9.0

305

1074

69

1.84

9.0

Applicant's summary and conclusion

Conclusions:
Based on all experiments performed for the determination of the rate of hydrolysis of E-BK105 at pH values normally found in the environment (pH 4-9), it was concluded that the half-life times of E-BK105 at 25°C are > 1 year at pH4, pH7 and pH9.