Registration Dossier

Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March 2010 - 08 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Principles of method if other than guideline:
In the course of the hydrolysis preliminary test (09/266-336ANE) 4,4-Diaminodiphenylether proved to be hydrolytically stable at pH 7 and 9; but significant decomposition of was observed at pH 4. Therefore the purpose of the tier 2 study is to perform the hydrolysis main test and evaluate the abiotic degradation of 4,4-Diaminodiphenylether at pH 4.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Remarks:
white
Radiolabelling:
no

Study design

Analytical monitoring:
no
Details on sampling:
Preliminary Test

Sterile aqueous buffer solutions of different pH values are treated with the test substance and incubated in the dark for 5 days. Concentration of the test substance is determined at the start and at the end of incubation. The reaction solutions were analysed at the start of the test and after five days with five replicate samples each.

Main Test:
At the end of the test three tubes were submitted for sterility confirmation. : Each analytical occasion and three vials were removed and analysed.
Buffers:
Preliminary Test:

pH 4.0: 5.11 g Potassium hydrogen phthalate was measured into a 500 ml volumetric flask and 1 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.

pH 7.0: 3.40 g Potassium dihydrogen phosphate was measured into a 500 ml volumetric flask and 73.9 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.

pH 9.0: 1.55 g Boric acid and 1.87 g Potassium chloride were measured into a 500 ml volumetric flask and 53.5 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water.

These sterile buffer solutions were prepared using reagent grade chemicals and ultra-pure, sterile water. The pH of each buffer solution was checked with a calibrated pH meter.

Main Test - pH 4 only.

6.80 g Potassium hydrogen phthalate was measured into a 1000 ml volumetric flask and 2 ml of 0.2 M Sodium hydroxide solution was added to it and was filled up to the mark with ultra-pure water. This sterile buffer solution was prepared using reagent grade chemicals and ultra-pure, sterile water.

Estimation method (if used):
None.
Details on test conditions:
PERFORMANCE OF THE TEST - Preliminary Test

Sterile aqueous buffer solutions of different pH values are treated with the test substance and incubated in the dark for 5 days. Concentration of the test substance is determined at the start and at the end of incubation.

Apparatus utilised:

HPLC system:
Merck-Hitachi LaChrom HPLC system:
D-7000 Interface, No.: 1231-056
L-7100 HPLC pump, No.: 1272-039
L-7200 Autosampler, No.: 1273-016
L-7400 UV Detector, No.: 1260-088
Jetstream II Plus Column Oven, No.: 160706
Balances: BP221S Sartorius, No.: 11809117; L2200P Sartorius, No.: 38100037
Thermostat: LP 132, No.: 870595
pH meter: METTLER TOLEDO DL 50 Graphix, No.: 5119033384
Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I
Hot Air Steriliser: ATP line FED, WTB Binder, No.: 9110-0035
Autoclave: LABOR MIM ST-174/6, No.: 4-3678
Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105
Centrifuge: Heraeus Biofuge, No.: 272369


Reagents and Materials utilised:

Methanol: HPLC grade, Carlo Erba, Lot No.: V9M706099M
Boric acid: GR for analysis, Merck, Batch No: A779365
Potassium dihydrogen phosphate: Analytical reagent grade, Reanal, Batch No.: KBR51912
Potassium chloride: GR for analysis, Merck, Batch No: K35661036
Potassium hydrogen phthalate: analytical reagent grade, Reanal, Batch No.: KBR51613
di-Potassium-hidrogen-phosphate: puriss, Reanal, Batch No.: KBM55318
Sodium hydroxide: analytical reagent grade, Reanal, Batch No.: KBM59615
Phosphoric Acid: for analysis, REANAL, Batch No.: KBM55463
Ultra pure water : ASTM Type I, prepared by Direct-Q 3 system, Millipore


Test solutions:
500 ml sterile solutions were prepared. The pH of each buffer solution was checked with a calibrated pH meter.

Storage of the solutions:
Solutions were transferred into 25 ml screw cap tubes. From each test solution seven tubes were prepared.

pH:
Hydrolysis was examined at three different pH values: 4.0, 7.0 and 9.0 in the dark.

Test temperature:
50 +/- 1.0 °C

Buffer solutions:
Composition is detailed above. These sterile buffer solutions were prepared using reagent grade chemicals and ultra-pure, sterile water.

The pH of each buffer solution was checked with a calibrated pH meter.

Light:
The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects.

Oxygen:
Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.

Sampling:
The reaction solutions were analysed at the start of the test and after five days with five replicate samples each.

Analysis of the samples:
Samples were diluted with methanol : water (1:1) mixture, then they were analysed with the HPLC method presented above.

Evaluation:
The chromatograms were evaluated with the help of “LaChrom Chromatogram Processor". Calculations were carried out using “EXCEL for Windows".

PERFORMANCE OF THE TEST - Main Test

Test solution:
Test item was dissolved in pH 4 buffer solution. The pH of the solution was checked with a calibrated pH meter.
Test solution was sterilised by filtering on a 0.22 µm Steritop filter unit.

Storage of the solutions:
Test solution was transferred into 20 ml headspace–vials under sterile circumstances, under a laminar flow hood. The tubes were entirely filled with the solution.
30 tubes of test solution and 10 tubes of control buffer were placed in each of the three thermostats (set to 25, 35 and 55 °C).

Sampling: Each analytical occasion and three vials were removed and analysed.
At the end of the test three tubes were submitted for sterility confirmation.

Analysis of the samples: Samples were diluted with methanol : water (1:1) mixture, then they were analysed with the above presented HPLC method.

Sterility confirmation:
At the end of the experiments three replicate samples of the test solutions from each temperature were submitted for sterility confirmation. The replicate samples were combined before the sterility testing. Samples were investigated using liquid culture media and the inoculated tubes were incubated at 30 °C for fourteen days. After the incubation period the tubes were evaluated for the growth of micro-organisms.

Test temperature:
25 +/- 0.5 °C, 35 +/- 0.5 °C and 55 +/- 0.5 °C

pH:
4.0

Light:
The hydrolysis reaction was carried out using dark thermostats to avoid photolytic effects.




Duration of testopen allclose all
Duration:
120 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
363.2 other: µg/mL
Remarks:
Preliminary test
Duration:
120 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
19.3 other: µg/mL
Remarks:
Preliminary test
Duration:
120 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
7.3 other: µg/mL
Remarks:
Preliminary test
Duration:
504 h
pH:
4
Temp.:
25 °C
Initial conc. measured:
309 other: µg/ml
Remarks:
Main test
Duration:
138 h
pH:
4
Temp.:
35 °C
Initial conc. measured:
309 other: µg/ml
Remarks:
Main test
Duration:
96 h
pH:
4
Temp.:
55 °C
Initial conc. measured:
186 other: µg/ml
Remarks:
Main test
Number of replicates:
Preliminary test: 5
Main test (pH-4): 5
Positive controls:
no
Negative controls:
no
Statistical methods:
The chromatograms were evaluated with the help of “LaChrom Chromatogram Processor". Calculations were carried out using “EXCEL for Windows".

Results and discussion

Preliminary study:
In the course of the preliminary test the observed hydrolysis of 4,4-Diaminodiphenylether was less than 10 per cent after 5 days at a temperature of 50 °C at pH 7 and 9. Therefore 4,4-Diaminodiphenylether is considered to be hydrolytically stable under these circumstances and normally no additional testing is required at pH 7 and 9.

Significant decomposition was observed at pH 4; 16 % of the original concentration was measured. Hence the higher tier test was performed at this pH value.
Test performance:
Preliminary test:
The pH of each buffer solution was checked with a calibrated pH meter.
The tubes were thermostated at 50 °C ± 1 °C.
The reaction solutions were analysed at the start of the test and after five days with five replicate samples each.
Calculations were carried out using “EXCEL for Windows".
Concentrations of the calibration samples were 0.02, 0.05, 0.2, 0.5, 2, 5 and 10 µg / ml. The correlation coefficient of each analytical occasion was 1.000.

Main test:
Test item was dissolved in pH 4 buffer solution. The pH of the solution was checked with a calibrated pH meter. Test solution was sterilised by filtering on a 0.22 µm Steritop filter unit.
Tubes of test solution and 10 tubes of control buffer were placed in each of the three thermostats (set to 25, 35 and 55 °C).
At the end of the experiments three replicate samples of the test solutions from each temperature were submitted for sterility confirmation. The replicate samples were combined before the sterility testing.
Calculations were carried out using “EXCEL for Windows" using linear regression.
The rate constants (kobs) of the tests were determined from the plots of the logarithms of the concentration versus time.
Concentrations of the calibration samples were 0.02, 0.05, 0.2, 0.5, 2, 5, and 10 µg / ml. The correlation coefficient of each analytical occasion was 1.000.
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
Not conducted
Total recovery of test substance (in %)open allclose all
% Recovery:
16
pH:
4
Temp.:
50 °C
Duration:
120 h
Remarks on result:
other: Preliminary test
% Recovery:
94
pH:
7
Temp.:
50 °C
Duration:
120 h
Remarks on result:
other: Preliminary test
% Recovery:
97
pH:
9
Temp.:
50 °C
Duration:
120 h
Remarks on result:
other: Preliminary test
% Recovery:
63
pH:
4
Temp.:
25 °C
Duration:
504 h
Remarks on result:
other: Main test
% Recovery:
61
pH:
4
Temp.:
35 °C
Duration:
138 h
Remarks on result:
other: Main test
% Recovery:
26
pH:
4
Temp.:
55 °C
Duration:
96 h
Remarks on result:
other: Main test
Dissipation DT50 of parent compoundopen allclose all
Key result
pH:
4
Temp.:
25 °C
DT50:
844 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
35 °C
DT50:
213 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
4
Temp.:
55 °C
DT50:
54 h
Type:
(pseudo-)first order (= half-life)
Details on results:
TEST CONDITIONS - Preliminary Test:
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): No

In the course of the preliminary test the observed hydrolysis of 4,4-Diaminodiphenylether was less than 10 per cent after 5 days at a temperature of 50 °C at pH 7 and 9. Therefore 4,4-Diaminodiphenylether is considered to be hydrolytically stable under these circumstances and normally no additional testing is required at pH 7 and 9.

Significant decomposition was observed at pH 4; 16 % of the original concentration was measured. Hence the higher tier test was performed at this pH value.

TEST CONDITIONS - Main Test (pH - 4):
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): No

Rate constants and half-lives measured at pH 4
25 °C, half life = 844 h (35 days)
35 °C, half life = 213 h (9 days)
55 °C, half life = 54 h (2 days)

Estimation of the half lives of the reaction at 20 and 25°C

The ln-transformed data of rate constants belonging to 25, 35 and 55 °C were plotted against 1/T (T= t+273.15 K). Based on the Arrhenius equation a line was fitted on the measured data and the rate constants were calculated for 25°C with the help of the equation of a regression line.
The calculated half-life of the reactions at 20 °C and 25 °C was determined to be 1177 hours and 714 hours respectively.

Results of the sterility confirmation test - main test: There were no microorganisms detected by the sterility confirmation test. (Ph.Hg.VIII – Ph.Eur. 6.3-1)

Any other information on results incl. tables

Results of the preliminary hydrolysis test

The hydrolysis test was performed at 50±0.5°C, at pH 4, 7 and 9. Measured concentrations and pH values are summarised in the table below: 

Measured data

pH

Sampling time, hour

Concentration of Test Item , µg/ml

Measured pH

Results of the separate test vessels

Mean withthe 95% confidence intervals

End/Start, %

4

0

(Start)

Control buffer

-

4.09

356.7

363.2 ± 5.5

-

4.10

360.9

364.4

366.2

367.7

120

Control buffer

-

4.00

56.7

57.5 ± 2.2

16

4.15

59.0

54.7

59.1

57.8

7

0

(Start)

Control buffer

-

7.02

19.1

19.3 ± 0.1

-

7.02

19.4

19.4

19.1

19.3

120

Control buffer

-

7.02

17.9

18.0 ± 0.1

94

7.04

18.0

18.0

18.0

18.1

9

0

(Start)

Control buffer

-

9.02

7.3

7.3 ± 0.1

-

9.04

7.4

7.4

7.3

7.3

120

Control buffer

-

9.09

7.1

7.1 ± 0.1

97

9.15

7.2

7.1

7.2

7.1

 

Results of the main hydrolysis test at pH = 4.

 

Temperature

Time

4,4-Diaminodiphenylether concentration

Measured pH

°C

hour

µg/ml

log value

% of the start

25

0

309

2.49

100

4.11

75

273

2.44

89

4.11

95

266

2.43

86

4.12

138

249

2.40

81

4.13

240

235

2.37

76

4.12

334

231

2.36

75

4.11

504

194

2.29

63

4.14

35

0

309

2.49

100

4.11

21.5

274

2.44

89

4.13

26

259

2.41

84

4.11

31

254

2.41

82

4.10

41.5

267

2.43

86

4.10

47

253

2.40

82

4.11

51

240

2.38

78

4.12

66

256

2.41

83

4.13

95

215

2.33

70

4.12

138

188

2.27

61

4.11

55

0

186

2.27

100

4.09

15.5

125

2.10

67

4.10

20

113

2.05

61

4.10

24.5

109

2.04

58

4.11

39.5

95

1.98

51

4.09

48.5

79

1.90

42

4.12

65.5

67

1.82

36

4.10

72.5

60

1.78

32

4.09

89

53

1.72

28

4.10

96

48

1.68

26

4.08

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The substance was deemed to be stable under the test conditions at pH 7 and 9. Significant decomposition was noted at pH4 in the preliminary as 16% only of the original test concentration was measured. A further test at pH 4 was therefore conducted at three temepratures (25, 35 and 55°C). At this pH, the following half lives were determined:
25 °C, half life = 844 h (35 days)
35 °C, half life = 213 h (9 days)
55 °C, half life = 54 h (2 days)
By extrapolation, the calculated half-life of the reactions at 20 °C and 25 °C was determined to be 1177 hours and 714 hours respectively.
Executive summary:

In the course of the preliminary test the observed hydrolysis of 4,4-Diaminodiphenylether was less than 10 per cent after 5 days at a temperature of 50 °C at pH 7 and 9. Therefore 4,4-Diaminodiphenylether is considered to be hydrolytically stable under these circumstances.

Significant decomposition was observed at pH 4. Therefore a main test was conducted at pH 4 and three temperatures (25, 35 and 55 °C). The calculated half-life of the reactions at 20 °C and 25 °C was determined to be1177 hours and 714 hours respectively.