1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

other information

Study period:

February 25-April 06, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study compliant with TG guideline and conducted under GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent: preliminary test: 0, 2.4 and 120 h at pH 4, pH 7 and pH 9; at pH 9 a main test (Tier 2) was performed with sampling intervals at 0 and 145 h (20 °C), 0, 53, 171, and 291 h (50 °C), 0, 3, 6, 24, 27, 31.5, and 47.75 h (60°C) and 0 and 6.25 h (70°C).
- Sampling method: samples were cooled to room-temperature prior analysis and diluted with 0.1% TFA in 50/50 (v/v) acetonitrile/water
- Sampling intervals/times for pH measurements: at each sampling interval
- Sample storage conditions before analysis: not indicated
- Other observation, if any (e.g.: precipitation, color change etc.): none

Buffers:

- pH: 4
- Type and final molarity of buffer: acetate buffer, 0.1 M
- Composition of buffer: 16.6% 0.1 M sodium acetate and 83.4% 0.1 M acetic acid containing 0.0009% (w/v) sodium azide
- pH: 7
- Type and final molarity of buffer: phosphate buffer, 0.1 M
- Composition of buffer: 0.1 M potassium dihydrogenphosphate adjusted to pH 7 using 10 N sodium hydroxide containing 0.0009% (w/v) sodium azide
- pH: 9
- Type and final molarity of buffer: borate buffer, 0.1 M
- Composition of buffer: 0.1 M boric acid and 0.1 M potassium chloride adjusted to pH 9 using 10 N sodium hydroxide containing 0.0009% (w/v) sodium azide

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: sterile glass or plastic vessels (depending on test temperature)
- Sterilisation method: solutions were filter-sterilised (0.2 µm FP 30/0.2 CA-S filter, Whatman, Dassel, Germany); buffer solutions contained
- Lighting: dark
- Measures to exclude oxygen: nitrogen gas was purged through the solutions for 5 minutes
- If no traps were used, is the test system closed/open: closed
- Is there any indication of the test material adsorbing to the walls of the test apparatus? no
TEST MEDIUM
- Volume used/treatment 6 ml
- Kind and purity of water: Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA)
- Preparation of test medium: Test substance solutions were prepared in the buffer solutions at a target concentration of 100 mg/I. 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.
- Renewal of test solution: none
- Identity and concentration of co-solvent: none

Duration of test

Duration of test 1

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

>= 85 - <= 90 mg/L

Duration of test 2

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

117 mg/L

Duration of test 3

Duration:

145 h

pH:

9

Temp.:

20 °C

Initial conc. measured:

>= 113 - <= 124 mg/L

Duration of test 4

Duration:

291 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

>= 110 - <= 116 mg/L

Duration of test 5

Duration:

47.75 h

pH:

9

Temp.:

60 °C

Initial conc. measured:

>= 109 - <= 113 mg/L

Duration of test 6

Duration:

6.25 h

pH:

9

Temp.:

70 °C

Initial conc. measured:

>= 98 - <= 102 mg/L

Number of replicates:

Preliminary test - Tier 1: duplicates
Mean study - Tier 2: triplicates

Positive controls:

no

Negative controls:

no

Statistical methods:

no statistical analysis performed

Results and discussion

Preliminary study:

In the preliminary test, 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, two preliminary tests were performed and in both tests a degree of hydrolysis of ≥ 10 % after 5 days was observed. According to the guideline, the higher Tier test was required to determine the half-life time of the test substance.
No test substance was detected in the blank buffer solutions.
The mean recoveries of the buffer solutions varied between 95 and 115%. At pH 4 and the repeated pH 9 test mean test recoveries fell within the criterion range of 90-110%. It demon-strated that the analytical method was adequate to support the hydrolysis study on the test substance. At pH 7 the mean recovery was 115%, thus slightly above the criterion range. However, this result was accepted since the analysed concentrations after 2.4 and 120 hours, respectively, were also above the nominal concentration level.
At pH 9, during the first test the mean recovery was higher than expected. A change in the colour of the test solution was observed in the 120-h samples. UV-Vis analysis revealed no shift of the absorbance maxima. An explanation for the increased recovery was not given. Yet, the results indicated a degree of hydrolysis of > 10% after 5 days. In order to confirm this result, the preliminary test at pH 9 was repeated. With a mean recovery at t=0 in the criterion range of 90-110%, the results of the second preliminary test were accepted.

Test performance:

In the main study at pH 9, at all temperatures tested, a decrease in concentration was observed with time. For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations were plotted against time. In case a linear relationship is obtained, the half-life times of the test substance can be determined according to the model for pseudo-first order reactions. However, when plotting concentration vs. time the observed curves were not in line with commonly found curves for hydrolysis, indicating that other effects than hydrolysis may be responsible for the decrease in analysed concentration. Therefore, at 50°C the hydrolysis behavior was after 171 hours again evaluated for 120 hours and since the decrease in concentration was< 10% it was concluded that the test substance was hydrolytically stable at pH 9. The results from the tests at 20°C and 60 °C confirm this finding.

Transformation products:

not measured

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

pH:

4

Temp.:

50 °C

DT50:

> 1 yr

Dissipation DT50 of parent compound 2

pH:

7

Temp.:

50 °C

DT50:

> 1 yr

Dissipation DT50 of parent compound 3

pH:

9

Temp.:

50 °C

DT50:

> 1 yr

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): At pH 9 at 50 °C within the first 7 days of exposure a decrease in the test substance concentration was observed which was assumed to not be due to hydrolysis but to other unknown effects. Therefore, hydrolysis was determined in the five days following the first seven days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Preliminary test: recoveries within 90-115%. The mean recovery of 115% at pH 7 was accepted since initially measured concentrations were also above the nominal concentration level. Main test: recoveries within 90-110%

Conclusions:

The test substance is hydrolytically stable.