Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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)

Version / remarks:

published in OJ No L 142/518 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

preliminary test: 0, 3, 4, 5 and 6 dayshydrolysis of unstable substances: 0, 0.97, 3.97, 5.97, 7.95, 11.94, 13.94, 19.96, 26.14 and 28.97 days

Estimation method (if used):

pH 4: 4 mL 0.1M NaOH + 500 mL 0.1M potassium hydrogen phthalate / 1 L (pH = 4.02 at 24°C)pH 7: 296 mL 0.1M NaOH + 500 mL 0.1M KH2PO4 / 1 L (pH = 7.08 at 23.6°C)pH 9: 213 mL 0.1M NaOH + 500 mL 0.1M H3BO3 in 0.1M KCl (pH = 9.08 at 23.7°C)

Details on test conditions:

PRELIMINARY TEST (Tier 1)The preliminary test is performed at 50 ± 0.5°C and pH 4.0, 7.0, and 9.0. If less than 10% of hydrolysis is observed after 5 days (t(½) > 1 year), the test substance is considered hydrolytically stable and, normally, no additional testing is required. If more than 10% of hydrolysis is observed within 5 days the test continues with Tire 2. If the substance is known to be unstable at environmentally relevant temperatures, the preliminary test is not required and the test starts with Tier 2 immediately. HYDROLYSIS OF UNSTABLE SUBSTANCES (Tier 2)Tier 2 is carried out at three temperatures from 10 to 70°C with following preferences for the first and second temperature: less than 25°C, and 50°C, respectively. Selected pH value results from performed preliminary test at which the test substance is found unstable. Each sample solution should be monitored at time intervals that provide a minimum of six spaced data points normally between 10 % and 90 % hydrolysis of the test substance. Duplicate analysis of the test substance for selected pH and temperature in separate reaction vessels is performed. Each test is stopped when 90% of hydrolysis is reached or after 30 days, whichever comes first.If the conditions of hydrolysis at the preliminary test fulfil above requirements, the results will be used for Tier 2 as they are. For each pH value and temperature at which Tier 2 is performed the linearity of data is statistically examined:- If linearity is found sufficient, pseudo first-order rate constant k(obs), half-life time and the parameters of Arrhenius equation are determined. The pseudo-first order rate constant of hydrolysis is estimated at 25°C and the test follows Tier 3.- If data show considerable non-linearity, the hydrolysis is not a first-order reaction and its rate constant, half-life time and the parameters of Arrhenius equation are not physically meaningful parameters. In this case, the value of DT50 is only determined by graphical interpolation (polynomial regression) for each pH / temperature and the test follows Tier 3.IDENTIFICATION OF HYDROLYSIS PRODUCTS (Tier 3)Any major hydrolysis products at least those representing ≥ 10% of the applied dose is identified by appropriate analytical methods. If appropriate, the calculations as described above are also applied to hydrolysis products.

Duration:

6 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

1 g/L

Duration:

6 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

1 g/L

Duration:

6 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

1 g/L

Duration:

29 d

pH:

4

Temp.:

22 °C

Initial conc. measured:

1 g/L

Duration:

29 d

pH:

4

Temp.:

35 °C

Initial conc. measured:

1 g/L

Duration:

29 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

1 g/L

Duration:

27 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

1 g/L

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Statistical methods:

For each pH value and temperature at which Tier 2 is performed the linearity of data is statistically examined:- If linearity is found sufficient, pseudo first-order rate constant k(obs), half-life time and the parameters of Arrhenius equation are determined. The pseudo-first order rate constant of hydrolysis is estimated at 25°C and the test follows Tier 3.- If data show considerable non-linearity, the hydrolysis is not a first-order reaction and its rate constant, half-life time and the parameters of Arrhenius equation are not physically meaningful parameters. In this case, the value of DT50 is only determined by graphical interpolation (polynomial regression) for each pH / temperature and the test follows Tier 3.

Preliminary study:

The preliminary test has shown that the test substance was not hydrolysed by more than 10% in period of 5 days at pH 7 and pH 9. Therefore, the test substance was considered to be hydrolytically stable at pH 7 and pH 9. It can be assumed that the half- time of hydrolysis at 25 °C would be approx. 1 year at pH 7 and pH 9. On the other hand the preliminary test has shown that the test substance was hydrolysed by more than 10% in period of 5 days at pH 4.

Test performance:

Tier 2 was carried out at pH4 at three temperatures 22°C, 35°C and 50 °C. The temperature of the thermostatic bath was controlled during the whole test when the sampling was done. A degree of hydrolysis (%) was calculated from the change of concentration. Each sample solution at pH 4 was measured at least six times during the hydrolysis. There was observation of degradation greater than 90 % at all temperatures over the period of 1 month. The test substance was considered to be unstable at pH 4.The results of Tier 2 have shown the linear dependence of logarithm of concentration on time. The hydrolysis was considered to be described as pseudo-first order reaction. The half-life times of hydrolysis of the test substance at 22°C, 35°C and 50°C in buffered solutions of nominal pH 4 were calculated.

Transformation products:

yes

No.:

#1

Key result

pH:

4

Temp.:

22 °C

Hydrolysis rate constant:

0.009 d-1

DT50:

80.5 d

Type:

(pseudo-)first order (= half-life)

Key result

pH:

4

Temp.:

35 °C

Hydrolysis rate constant:

0.024 d-1

DT50:

29.02 d

Type:

(pseudo-)first order (= half-life)

Key result

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.09 d-1

DT50:

7.72 d

Type:

(pseudo-)first order (= half-life)

Key result

pH:

7

Temp.:

50 °C

DT50:

> 1 yr

Key result

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): Seven following sample solutions in buffer pH 4 used for Tier 2 were tested for sterility after hydrolysis test: solutions I and II at 22°C, solutions I and II at 35°C, solutions I and II at 50°C (Tier 2 as extension of Tier 1) and solution II at 50°C (new Tier 2). Solution I at 50°C (new Tier 2) couldn´t be tested for sterility due to damage during hydrolysis test. The sterility of first six solutions was confirmed. The sterility of the last mentioned solution was not confirmed, which has no influence on the results of HPLC-MS identification (compare chromatograms with confirmed sterility). The solution II at 50°C (new Tier 2) was used for Tier 3 and the sterility test was done afterwards. Unsterility of this solution was probably caused by contamination during manipulation at Tier 3. MAJOR TRANSFORMATION PRODUCTS At pH 4: Identified the hydrolytic product of the Reactive Orange 13. At pH 7: The test substance is stable at this pH. At pH 9: The test substance is stable at this pH.

Table 1: Preliminary test

Day	Hydrolysis (%)
	pH 4.03 at 50°C		pH 7.05 at 50°C		pH 8.96 at 50°C
	1	2	1	2	1	2
0 (start)	0.0	0.0	0.0	0.0	0.0	0.0
3	23.9	23.9	0.7	0.2	2.7	3.0
4	30.5	30.5	0.5	0.2	3.9	4.0
5	36.7	36.6	0.5	0.2	4.9	4.8
6	42.3	42.1	0.4	0.2	5.8	5.8
Average of hydrolysis (%)	42.2		0.3		5.8

Table 2: Hydrolysis of unstable substances

Time [days]	Temperature [°C]	Hydrolysis [%]
0 - start	22.0	0.0	0.0
0.97	22.0	0.7	0.9
3.97	22.2	3.1	3.3
5.97	22.0	4.9	5.1
7.95	22.1	6.4	6.7
11.94	22.2	9.3	9.6
13.94	22.0	11.1	11.3
19.96	22.0	15.7	15.9
26.14	22.0	20.3	20.3
28.97	22.0	21.8	21.9
Time [days]	Temperature [°C]	Hydrolysis [%]
0.00	35.0	0.0	0.0
0.97	35.1	2.7	2.7
3.97	35.1	10.3	10.4
5.97	35.2	15.3	15.2
7.95	35.1	19.4	19.4
11.94	35.1	27.6	27.8
13.94	35.2	31.6	31.1
19.96	35.1	39.0	39.1
25.96	35.1	45.9	46.7
28.97	35.0	50.5	50.5
Time [days]	Temperature [°C]	Hydrolysis [%]
0.00	49.6	0.0	0.0
3.00	49.7	23.9	23.9
4.01	49.7	30.5	30.5
5.01	49.7	36.7	36.6
6.00	49.6	42.3	42.1
13.20	49.6	69.5	69.5
14.09	49.7	72.1	72.1
17.09	49.6	78.6	78.5
19.09	49.7	82.1	82.2
21.06	49.6	85.0	84.9
25.06	49.7	89.6	89.5
27.06	49.7	91.2	91.2

Validity criteria fulfilled:

yes

Conclusions:

The preliminary test has shown that the test substance was not hydrolysed by more than 10 % in period of 5 days at pH 7 and pH 9. Therefore, the test substance was considered to be hydrolytically stable at pH 7 and pH 9. It can be assumed that the half- time of hydrolysis at 25°C would be approx. 1 year at pH 7 and pH 9. On the other hand the preliminary test has shown that the test substance was hydrolysed by more than 10 % in period of 5 days at pH 4. Due to hydrolytic instability at pH 4 the testing at pH 4 continued to Tier 2 (Hydrolysis of unstable substances), Tier 3 (Identification of hydrolysis product) and sterility confirmation test at the end of the hydrolysis. Tier 2 was carried out at pH4 at three temperatures 22°C, 35°C and 50 °C. The temperature of the thermostatic bath was controlled during the whole test when the sampling was done. A degree of hydrolysis (%) was calculated from the change of concentration. Each sample solution at pH 4 was measured at least six times during the hydrolysis. There was observation of degradation greater than 90 % at all temperatures over the period of 1 month. The test substance was considered to be unstable at pH 4.The results of Tier 2 have shown the linear dependence of logarithm of concentration on time. The hydrolysis was considered to be described as pseudo-first order reaction. The half-life times of hydrolysis of the test substance at 22°C, 35°C and 50°C in buffered solutions of nominal pH 4 were calculated and the results of Tier 2 are presented in the following tables.

Executive summary:

The test of hydrolysis as a function of pH for Reactive Orange 13 was performed. The procedure was performed according to Method C.7 – Degradation – Abiotic Degradation: Hydrolysis as a Function of pH, Council regulation (EC) No 440/2008. Published in OJ No L 142/518 May 2008 and OECD Test Guideline No.111, Hydrolysis as a function of pH, Adopted April 13, 2004.

The preliminary test has shown that the test substance was not hydrolysed by more than 10 % in period of 5 days at pH 7 and pH 9. Therefore, the test substance was considered to be hydrolytically stable at pH 7 and pH 9. It can be assumed that the half-time of hydrolysis at 25 °C would be approximately 1 year at pH 7 and pH 9.

On the other hand the preliminary test has shown that the test substance was hydrolysed by more than 10 % in period of 5 days at pH 4.

Due to hydrolytic instability at pH 4 the testing at pH 4 continued to Tier 2 (Hydrolysis of unstable substances) and consequently to Tier 3 (Identification of hydrolysis product) and sterility confirmation test at the end of the hydrolysis.

The results of Tier 2 have shown the linear dependence of logarithm of concentration on time. The hydrolysis was considered to be described as pseudo-first order reaction.

The half-life times of hydrolysis of the test substance at 22°C, 35°C and 50°C in buffered solutions of nominal pH 4 were calculated and the results are presented in the following table.

The test substance at pH 4 is hydrolytically unstable and according to hydrolysis test, Tier 2 the half-life time of hydrolysis at 25°C was estimated at 64 days.

Tier 3 (Identification of hydrolysis product) was performed by HPLC-UV-MS. Hydroxy-derivatives of Rreactive Orange 13 as hydrolytic products (Compound 1B with RT=2.1 min and Compound 2 with RT=5.4 min), RO13 (Compound 3 with RT=8.2 min) and phtalic acid (Compound 1A with RT=2.1 min) were identified at the sample solution pH 4.

Sterility confirmation test was performed with each sample solution at the end of the hydrolysis at conditions 72h / 37°C. Sterility of solutions was confirmed.

Key value for chemical safety assessment

Half-life for hydrolysis:

63.9 d

at the temperature of:

25 °C