Mixture of hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-5-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate and hexa(sodium,lithium) 4-{[1,7-disulfonato-5-hydroxy-6-({4-[(6-methoxy-7-sulfonatobenzoheteromonocycle-2-yl)diazenyl]-5-alkyl-2-(3-sulfonatopropoxy)phenyl}diazenyl)naphthalen-2-yl]diazenyl}-5-hydroxy-1-(4-sulfonatophenyl)heteromonocycle-3-carboxylate

Hydrolysis

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 guideline

Test guideline 1

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.

Test guideline 2

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

Test material information

Constituent 1

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 test

Duration of test 1

Duration:

5 d

pH:

4

Initial conc. measured:

74.3 - 76.2 mg/L

Duration of test 2

Duration:

5 d

pH:

7

Initial conc. measured:

109 - 113 mg/L

Duration of test 3

Duration:

5 d

pH:

9

Initial conc. measured:

101 - 102 mg/L

Duration of test 4

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 %)

Total recovery of test substance (in %) 1

% Recovery:

74 - 76

pH:

4

Temp.:

49.9 °C

Total recovery of test substance (in %) 2

% Recovery:

105 - 109

pH:

7

Temp.:

49.9 °C

Total recovery of test substance (in %) 3

% 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 test–hydrolysis 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 test–hydrolysis 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.