Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Performance of the Preliminary Test

Fortified Samples:
(90 % Level) A stock solution of the test item was prepared in pure water at a concentration level of approx. 1 g/L. 2.25 mL of the stock solution were transferred into a 25 mL volumetric flask and filled up to the mark with the respective buffer solution resulting in a final concentration of about 90 mg/L. Five replicates were prepared for each pH-value. The fortified samples were treated as the test solution meaning that the samples were first filled into 10 mL volumetric flasks and thereafter diluted 1:1 v/v with acetonitrile.

Test Solution:
A stock solution of the test item was prepared in pure water at a concentration level of approx. 10 g/L. 2 mL of the stock solution were transferred into a 200 mL volumetric flask and filled up to the mark with the respective buffer solution resulting in a final concentration of about 100 mg/L. The concentration was below 0.01 M or half of the saturation concentration of the test item in water.

Incubation of the Test Solution:
The solutions were incubated at approx. 50 °C at three different pH values (4, 7, 9) at time intervals from 0 h to 120 h.
Sampling: Three samples of solutions of each pH-value were taken after 0 h for the determination of the applied concentration whereas two samples were taken at each following sampling point.
Sampling points: 0, 4, 24, 120 hours.
Sample Preparation: After sampling, samples were diluted 1:1 v/v with acetonitrile to stop the hydrolysis process.
Sterility Control: A sterility confirmation test was performed at the end of the incubation period. To do this, separate test vessels were incubated under test conditions as described above. At the end of the maximal incubation period dip slides for fluids (Roti-Dip Slides (PCA/RBCplus), Carl-Roth GmbH, Karlsruhe, Germany) were wetted completely with the samples. Excess sample was allowed to run off. The dip slides were placed back into their tube and incubated at 21 ± 1 °C for 4 days. No colonies on the agar were observed.

Performance of the Main Test at pH 4
According to the results of the preliminary test a decline of the test item >10 % of the nominal concentration was found at pH 4 after 5 days at 50 °C. Thus, a main test at this specific pH had to be performed.

Test Solution:
A stock solution of the test item was prepared in pure water at a concentration level of approx. 10 g/L. 2.0 or 2.5 mL of the stock solution were transferred into a 200 or 250 mL volumetric flask and filled up to the mark with the respective buffer solution resulting in a final concentration of about 100 mg/L. The concentration was below 0.01 M or half of the saturation concentration of the test item in water.

Fortified Samples: (10 % Level)
A stock solution of the test item was prepared in pure water at a concentration level of approx. 1 g/L. The stock solution was diluted with the respective buffer solution resulting in a final concentration of about 10 mg/L. Five replicates were prepared at a pH of 4. The fortified samples were treated as the test solution meaning that the samples were first filled into 10 mL volumetric flasks and thereafter diluted 1:1 v/v with acetonitrile.

Performance of the Main Test pH 4 at 20 °C
Incubation of the Test Solution: The test item solution was incubated at 20 ± 2.0 °C in the dark.
Sampling: Three samples were taken after 0 d for the determination of the applied concentration whereas two samples were taken at each following sampling point.
Sampling points: 0, 4, 8, 11, 15, 18, 22, 25, 29 days

Test Duration:
Maximum incubation time was 29 days.

Sample Preparation:
After sampling, samples were diluted 1:1 v/v with acetonitrile to stop the hydrolysis process.

Sterility Control:
A sterility confirmation test was performed at the end of the incubation period. To do this, separate test vessels were incubated under test conditions as described above. At the end of the maximal incubation period dip slides for fluids (Hycon GK-T/HS, Heipha Dr. Müller GmbH, Eppelheim, Germany) were wetted completely with the samples. Excess sample was allowed to run off. The dip slides were placed back into their tube and incubated at 21 ± 1 °C for 4 days. No colonies on the agar were observed.

Documentation:
All observations and the measurements obtained were documented in the raw data for each experiment.

Performance of the Main Test pH 4 at 50 °C

Incubation of the Test Solution:
The test item solution was incubated at 50 °C ± 0.5 °C in the dark.

Sampling:
Three samples were taken after 0 d for the determination of the applied concentration whereas two samples were taken at each following sampling point.

Sampling points: 0, 4, 8, 11, 15, 18, 22, 25, 29 days

Test Duration:
Maximum incubation time was 29 days.
Sample Preparation: After sampling, samples were diluted 1:1 v/v with acetonitrile to stop the hydrolysis process.
Sterility Control: A sterility confirmation test was performed at the end of the incubation period. To do this, separate test vessels were incubated under test conditions as described above. At the end of the maximal incubation period dip slides for fluids (Hycon GK-T/HS, Heipha Dr. Müller GmbH, Eppelheim, Germany) were wetted completely with the samples. Excess sample was allowed to run off. The dip slides were placed back into their tube and incubated at 21°C ± 1°C for 4 days. No colonies on the agar were observed.
Documentation: All observations and the measurements obtained were documented in the raw data for each experiment.

Performance of the Main Test pH 4 at 60 °C
Incubation of the Test Solution: The test item solution was incubated at 60 ± 5.7 °C.
Sampling: Three samples were taken after 0 d for the determination of the applied concentration whereas two samples were taken at each following sampling point.
Sampling points: 0, 2, 5, 7, 9, 12, 14 days
Test Duration: Maximum incubation time was 14 days.

Sample Preparation:
After sampling, samples were diluted 1:1 v/v with acetonitrile to stop the hydrolysis process.

Sterility Control:
A sterility confirmation test was performed at the end of the incubation period. To do this, separate test vessels were incubated under test conditions as described above. At the end of the maximal incubation period dip slides for fluids (Roti-Dip Slides (PCA/RBCplus), Carl-Roth GmbH, Karlsruhe, Germany) were wetted completely with the samples. Excess sample was allowed to run off. The dip slides were placed back into their tube and incubated at 21 ± 1°C for 4 to 5 days. No colonies on the agar were observed.

Documentation:
All observations and the measurements obtained were documented in the raw data for each experiment.

Buffers:

The buffer solutions were prepared as described below. The pH of each buffer solution at the relevant temperatures was checked with a pH meter. Each buffer solution was purged with nitrogen gas and sterilized by heating in an autoclave before using it to prepare the test solution.

Pre-Test:
pH 4:
C8H5KO4 buffer (0.1 M)
500 mL potassium hydrogen phthalate solution (10.212 g C8H5KO4 / 500 mL pure water) was mixed with 40 mL sodium hydroxide solution (0.1 mol/L NaOH) and filled up to 1000 mL with pure water resulting in a pH-value of 4.0 (at 21 °C).

pH 7:
KH2PO4 buffer (0.1 M)
500 mL potassium dihydrogen phosphate solution (6.082 g KH2PO4 / 500 mL pure water) were mixed with 296 mL sodium hydroxide solution (0.1 mol/L NaOH) and filled up to 1000 mL with pure water resulting in a pH-value of 7.0 (at 22 °C).

pH 9:
Boric acid buffer (0.1 M)
500 mL boric acid solution (3.082 g H3BO3 / 500 mL KCl 0.1 M) was mixed with 213 mL sodium hydroxide solution (0.1 M NaOH) and filled up to 1000 mL with pure water resulting in a pH-value of 9.0 (at 22 °C).

Main Test:
Citric acid buffer (0.1 M)
330 mL citric acid solution (21.013 g citric acid / 1000 mL pure water) were mixed with 170 mL sodium citrate solution (29.409 g C6H5O7Na3 x 2 H2O / 1000 mL pure water) and filled up to 1000 mL with pure water resulting in a pH-value of 4.0 (at 20 °C).
The buffer solution was purged with nitrogen gas and sterilized by heating in an autoclave before using it to prepare the test solution.

Details on test conditions:

Surrounding Conditions: The test containers were maintained at constant temperature in the dark:


Surrounding Conditions: The vessels containing the test solution were maintained at constant temperature in the dark:
Pre-test (pH 4, 7, 9):
50°C ± 0.5°C, darkness
Main-test (pH 4):
Temp. A: 20°C ± 2.0°C, darkness
Temp. B: 50°C ± 0.5°C, darkness
Temp. C: 60°C ± 5.7°C, darkness

Test Vessels: Stoppered glass flasks were used for carrying out the tests. All glassware was sterilised before usage.
Reagents: Purity ≥ 99%

Duration:

29 d

pH:

4

Temp.:

20 °C

Initial conc. measured:

100.79 mg/L

Duration:

29 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

100.99 mg/L

Duration:

14 d

pH:

4

Temp.:

60 °C

Initial conc. measured:

98.33 mg/L

Number of replicates:

Two samples of solutions of each pH value at each test temperature were taken at each sampling point.

Positive controls:

no

Negative controls:

no

Statistical methods:

None

Preliminary study:

The test item was dissolved in aqueous solutions buffered at pH 4, 7 and 9 and incubated at 50 ± 2.4 °C for maximum of 5 days.
In the incubated samples at pH 7 and 9 no significant decline of the test item concentration was observed. After 5 days of incubation more than 90 % of the initial concentrations could be observed.
No main test was to be performed at pH 7 and 9.
In case of samples incubated at pH 4 (phthalate buffer) a significant degradation of the test item was observed. After 5 days of incubation 75 % of the initial concentration could be observed.
A main test was to be perfomed at pH 4.

Transformation products:

not measured

% Recovery:

98

St. dev.:

0

pH:

4

Temp.:

20 °C

Duration:

29 d

% Recovery:

89

St. dev.:

0

pH:

4

Temp.:

50 °C

Duration:

29 d

% Recovery:

91

St. dev.:

1

pH:

4

Temp.:

60 °C

Duration:

14 d

Remarks on result:

hydrolytically stable based on preliminary test

Details on results:

Samples at pH 4 were incubated at 20, 50 and 60°C for 29 d and 14 d, respectively. The buffer solution was changed to reduce matrix effects and to enhance the sensitvity of the analytical method.
In the incubated samples no significant decline of the test item concentration was observed. At the end of incubation at least 89 % of the initial concentration could be found. Under environmental relevant conditions (20 °C) 98 % of the initial amount were detected after 29 days of incubation.
As the test item was found to be stable further calculations (e.g. reaction rate constant or half-life) were not executed.

Results of the Preliminary Tests

		Concentration in Samples			Recovery
Incubation Period	Replicate	Found	Dilution Factor	Calculated^1	% Nominal	Mean
[h]		[mg/L]		[mg/L]
pH 4
0	A	44.334	2	88.669	101 %	100 %
	B	43.693	2	87.386	100 %
	C	43.561	2	87.123	99 %
4	A	45.812	2	91.625	104 %	104 %
	B	45.727	2	91.454	104 %
24	A	43.275	2	86.550	99 %	99%
	B	43.150	2	86.301	98 %
120	A	32.018	2	64.036	73 %	75 %
	B	33.454	2	66.908	76 %
pH 7
0	A	52.431	2	104.863	100 %	100 %
	B	52.430	2	104.860	100 %
	C	52.483	2	104.966	100 %
4	A	52.993	2	105.986	101 %	102%
	B	53.460	2	106.919	102 %
24	A	52.537	2	105.073	100 %	103%
	B	54.977	2	109.954	105 %
120	A	51.115	2	102.230	97 %	98 %
	B	51.388	2	102.776	98 %
pH 9
0	A	52.358	2	104.716	100 %	100 %
	B	52.331	2	104.662	100 %
	C	52.126	2	104.253	100 %
4	A	52.595	2	105.190	101 %	100 %
	B	52.281	2	104.562	100 %
24	A	56.366	2	112.732	108 %	109 %
	B	57.055	2	114.111	109 %
120	A	50.826	2	101.651	97 %	96%
	B	48.982	2	97.965	94 %
^1Values calculated from exact raw data
Nominal concentrations: pH4 = 87.73 mg/L; pH7 = 104.90 mg/L; pH9 = 104.54 mg/L

Results of the Main Test

		Concentration in Samples			Recovery
Incubation Period	Replicate	Found	Dilution Factor	Calculated^1	% Nominal	Mean
[d]		[mg/L]		[mg/L]
20°C
0	A	50.956	2	101.912	101 %	100 %
	B	50.218	2	100.435	100 %
	C	50.009	2	100.017	99 %
4	A	47.977	2	95.954	95 %	96 %
	B	48.510	2	97.021	96 %
8	A	48.510	2	97.020	96 %	97 %
	B	48.764	2	97.528	97 %
11	A	51.673	2	103.345	103 %	103 %
	B	51.803	2	103.606	103 %
15	A	50.006	2	100.011	99 %	99 %
	B	49.620	2	99.240	98 %
18	A	48.634	2	97.268	97 %	98 %
	B	49.641	2	99.282	99 %
22	A	49.408	2	98.817	98 %	98 %
	B	49.183	2	98.366	98 %
25	A	50.485	2	100.971	100 %	99 %
	B	49.044	2	98.087	97 %
29	A	49.547	2	99.095	98 %	98 %
	B	49.517	2	99.034	98 %
50°C
0	A	50.288	2	100.575	100 %	100 %
	B	50.454	2	100.908	100 %
	C	50.748	2	101.495	100 %
4	A	47.417	2	94.835	94 %	94%
	B	47.012	2	94.024	93 %
8	A	47.540	2	95.080	94 %	94%
	B	47.054	2	94.107	93 %
11	A	49.884	2	99.768	99 %	98%
	B	49.234	2	98.469	98 %
15	A	47.649	2	95.299	94 %	95 %
	B	48.553	2	97.105	96 %
18	A	46.313	2	92.627	92 %	92 %
	B	46.882	2	93.764	93 %
22	A	46.718	2	93.437	93 %	93%
	B	47.590	2	95.179	94 %
25	A	46.374	2	92.748	92 %	92 %
	B	46.600	2	93.201	92 %
29	A	44.951	2	89.901	89 %	89 %
	B	44.797	2	89.595	89 %
60°C
0	A	48.957	2	97.914	100 %	100 %
	B	49.216	2	98.433	100 %
	C	49.321	2	98.643	100 %
2	A	47.416	2	94.831	96 %	96 %
	B	46.893	2	93.785	95 %
5	A	49.597	2	99.194	101 %	101%
	B	49.706	2	99.412	101 %
7	A	48.133	2	96.266	98 %	99 %
	B	49.088	2	98.177	100 %
9	A	47.047	2	94.095	96 %	95 %
	B	46.580	2	93.160	95 %
12	A	45.482	2	90.963	93 %	92%
	B	44.538	2	89.075	91 %
14	A	45.341	2	90.682	92 %	91 %
	B	44.519	2	89.038	91 %
^1Values calculated from exact raw data
Nominal concentrations: 20 °C = 100.79 mg/L; 50 °C =100.99 mg/L; 60 °C = 98.33 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The test item was hydrolytically stable at pH 4, 7 and 9.

Executive summary:

The abiotic degradation (Hydrolysis as a function of pH)

of the test item (FAT 40210/F TE)was determined based on the procedures indicated by the following internationally accepted methods:

- OECD Guideline No. 111

- EU Method C.7

Pre-Test:

The test item was dissolved in aqueous solutions buffered at pH 4, 7 and 9 and incubated at 50 ± 2.4 °C for maximum of 5 days.

In the incubated samples at pH 7 and 9 no significant decline of the test item concentration was observed. After 5 days of incubation more than 90% of the initial concentrations could be observed.

No main test was to be performed at pH 7 and 9.

In case of samples incubated at pH 4 (phthalate buffer) a significant degradation of the test item was observed. After 5 days of incubation 75 % of the initial concentration could be observed.

A main test was to be perfomed at pH 4.

Main Test:

Samples at pH 4 were incubated at 20, 50 and 60 °C for 29 d and 14 d, respectively. The buffer solution was changed to reduce matrix effects and to enhance the sensitvity of the analytical method.

In the incubated samples no significant decline of the test item concentration was observed. At the end of incubation at least 89 % of the initial concentration could be found. Under environmental relevant conditions (20 °C) 98 % of the initial amount were detected after 29 days of incubation.

As the test item was found to be stable further calculations (e.g. reaction rate constant or half-life) were not executed. 

This study is classified acceptable and satisfies the guideline requirements for hydrolysis studies.

Description of key information

Neither degradation rate nor corresponding half life values were calculated as the test item was found to be stable at pH 4, 7 and 9.

Key value for chemical safety assessment

Half-life for hydrolysis:

6.7 yr

at the temperature of:

20 °C

Additional information

In a GLP compliant OECD 111 guideline study the test substance is found to be hydrolytically stable at pH 4, 7 and 9. In this study, in the incubated samples no significant decline of the test item concentration was observed. At the end of incubation at least 89 % of the initial concentration could be found. Under environmental relevant conditions (20 °C) 98 % of the initial amount were detected after 29 days of incubation. As the test item was found to be stable further calculations (e.g. reaction rate constant or half-life) were not executed.