Trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 January 2020 to 6 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test sample was corrected for purity of the test material.

Radiolabelling:

no

Analytical monitoring:

yes

Remarks:

High-performance liquid chromatography

Details on sampling:

At initiation, one test solution was pretreated and analysed. The remaining test solutions were put into the test equipment and warmed at the test temperature.
After 5 days of Tier 1 or at the measurement point of Tier 2, two test solutions were taken out from test equipment and were cooled to room temperature, and then were pretreated and analysed.

Buffers:

- pH: 4.0
- Preparation of buffer: The solution was prepared by mixing 4.5 mL of 1 mol/L sodium hydroxide solution and 50 mL of 0.5 mol/L potassium dihydrogen citrate solution, and then filling up to 500 mL with purified water, and then adjusting to pH 4.0 with 1 mol/L hydrochloric acid.

- pH: 7.0
- Preparation of buffer: The solution was prepared by mixing 14.8 mL of 1 mol/L sodium hydroxide solution and 50 mL of 0.5 mol/L potassium dihydrogenphosphate solution, and then filling up to 500 mL with purified water.

- pH: 9.0
- Preparation of buffer: The solution was prepared by mixing 10.7 mL of 1 mol/L sodium hydroxide solution and 50 mL of 0.5 mol/L potassium chloride and 0.5 mol/L boric acid solution, and then filling up to 500 mL with purified water.

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 mL test tube with glass stopper
- Lighting: Protection from light (test vessels were covered with aluminum foil)
- Confirmation of sterile conditions: Sterile conditions were confirmed for one of the test solutions at the final measurement point of each test temperature at pH 4.0 according to the plate culture method using agar medium.

TEST MEDIUM
- Volume used/treatment: ca. 10 mL
- Kind and purity of water: purified water (not further specified)
- Preparation of test medium:
The test sample (200.09 mg) was precisely weighed with an electronic analytical balance and dissolved in purified water with ultrasonic irradiation, and then filled up to 10 mL with purified water to obtain 16600 mg/L test material solution. The pH of the test solution was measured.
A portion (2 mL, 33.3 mg as test material) of the test material solution was put into a 200 mL volumetric flask and then filled up with each buffer solution. This solution was aerated for about 5 minutes with helium gas after filtration with a sterile filter (0.22 µm) to obtain a test solution.
The test solution (about 10 mL) was poured into respective test vessels and the test vessels were sealed.

PRELIMINARY TEST
In a preliminaly test, the test material was not hydrolysed at pH 7.0 and pH 9.0, but was hydrolysed at pH 4.0. Therefore, in the main study, the OECD 111 Tier 1 test was performed at pH 7.0 and pH 9 .0, and Tier 2 and Tier 3 tests were performed at pH 4.0.

CALCULATION OF RESULTS
- Calculation for residual percentage of test material (Tier 1)
The residual percentage of test material at pH7.0 and pH 9.0 in Tier 1 was calculated from the following equation:
Residual percentage of test material (%) = (C / Ci) x 100
where:
Ci : Concentration of test material at initiation (mg/L)
C : Concentration of test material after 5 days (mg/L)
In case that the residual percentage of test material is larger than 90%, the test material is judged to be hydrolytically stable.
- Calculation ofhydrolysis rate constant and the half life of test material (Tier 2)
A regression equation was obtained by plotting logarithm of the test material concentration at each time (log C) versus time (t), and then slope (a) and intercept (b) were calculated at pH 4.0.
The regression equation is shown as follows: logC = at + b
The hydrolysis rate constant of test material and the half life were calculated by the following equation:
k = -2.303 x a
t1/2= 0.693 / k
where
k: hydrolysis rate constant
t1/2: half life
Logarithm values of the hydrolysis rate constants were plotted versus reciprocal number of the respective absolute temperature (1/T) and a regression equation was obtained by the method of least squares. The hydrolysis rate constant and the half life at 25°C were calculated by extrapolation from the regression equation.

Duration:

5 d

pH:

7.04

Temp.:

50 °C

Initial conc. measured:

168 mg/L

Remarks:

Tier 1 Test

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

165 mg/L

Remarks:

Tier 1 Test

Duration:

22 d

pH:

4.02

Temp.:

50 °C

Initial conc. measured:

165 mg/L

Remarks:

Tier 2 Test

Duration:

10 d

pH:

4.03

Temp.:

60 °C

Initial conc. measured:

167 mg/L

Remarks:

Tier 2 Test

Duration:

4 d

pH:

4.02

Temp.:

70 °C

Initial conc. measured:

166 mg/L

Number of replicates:

Single measurement at test initiation. Duplicate measurements after 5 days (Tier 1) or at the measuring point (Tier 2).

Positive controls:

not specified

Negative controls:

not specified

Statistical methods:

not specified

Preliminary study:

In a preliminaly test, the test material was not hydrolysed at pH 7.0 and pH 9.0, but was hydrolysed at pH 4.0.

Transformation products:

not specified

Remarks:

Please refer to 'Any other information on results inc. tables'

Details on hydrolysis and appearance of transformation product(s):

Please refer to 'Any other information on results inc. tables'

% Recovery:

99.4

pH:

7

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

% Recovery:

93.9

pH:

9

Temp.:

50 °C

Duration:

5 d

Remarks on result:

hydrolytically stable based on preliminary test

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.003 h-1

DT50:

229 h

Type:

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

Remarks on result:

other: average values of two measurements

pH:

4

Temp.:

60 °C

Hydrolysis rate constant:

0.007 h-1

DT50:

94.2 h

Type:

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

Remarks on result:

other: average values of two measurements

pH:

4

Temp.:

70 °C

Hydrolysis rate constant:

0.015 h-1

DT50:

46.3 h

Type:

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

Remarks on result:

other: average values of two measurements

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

93.6 d

Type:

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

Remarks on result:

other: extrapolated value

Details on results:

The residual percentage of test material at pH 7.0 and pH 9 .0 was larger than 90% in Tier 1. Therefore, the test material can be considered hydrolytically stable at pH 7.0 and pH 9 .0.
In Tier 2 at pH 4.0, the hydrolysis rate constant and the half life at 25°C are considered to be acceptable because the regression equation about logarithm values of the hydrolysis rate constants versus reciprocal number of the respective absolute temperature (1/T) was confirmed to be a straight line. As the colony was not counted after the test (number of colonies counted at pH 4.0, at 50, 60 and 70°C was zero), it was considered that sterile conditions were maintained. Therefore, microoganism was not affected the production of hydrolysis products.

Identification of hydrolysis products

Name	Retention time	Detection ion (m/z) and attribution	Presumed molecular weight
Hydrolysis product-1	About 0.4	191.0: Unknown 390.9: Unknown	Unknown
Hydrolysis product-2	About 7.3/7.4	350.5: [M2-2H]^2- 702.1: [M2-H]^-	M2^*1: 703
Test material	About 9.7	359.6: [M-3Na+H]^2- 720.0/722.0: [M-3Na+2H]^-	M: 787/789

^*1 M2: [M-3Na+3H-Cl-+OH]

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the Tier 1 study, the test material was determined to be hydrolytically stable at pH 7 and pH 9. The test material was not found to be hydrolytically stable at pH 4. In Tier 2, the hydrolysis rate constant of the test material was determined to be 3.09 x 10^-4 hour^-1 with an associated half-life of 93.6 days, at 25°C, pH 4.

Executive summary:

The hydrolysis of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 111, and under GLP conditions.

Under the conditions of the Tier 1 study, the test material was determined to be hydrolytically stable at pH 7 and pH 9. The test material was not found to be hydrolytically stable at pH 4. In Tier 2, the hydrolysis rate constant of the test material was determined to be 3.09 x 10^-4 hour^-1 with an associated half-life of 93.6 days, at 25°C, pH 4.

Description of key information

The test material was determined to be hydrolytically stable at pH 7 and pH 9.

At pH 4 (25°C), the hydrolysis rate constant of the test material was determined to be 3.09 x 10^-4 hour^-1 with an associated half-life of 93.6 days.

Key value for chemical safety assessment

Half-life for hydrolysis:

93.6 d

at the temperature of:

25 °C

Additional information

The hydrolysis of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 111, and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Under the conditions of the Tier 1 study, the test material was determined to be hydrolytically stable at pH 7 and pH 9. The test material was not found to be hydrolytically stable at pH 4. In Tier 2, the hydrolysis rate constant of the test material was determined to be 3.09 x 10^-4 hour^-1 with an associated half-life of 93.6 days, at 25°C, pH 4.