Reaction products of 1-(substitutedphenyl)urea coupled with diazotated potassium sodium substituted-5-{[2-(substituted)ethyl]sulfonyl}benzenesulfonate, further condensed with 2,4,6-trichloro-1,3,5-triazine, further converted with disubstituted benzene-1,4-disulfonic acid in aq. sodium hydroxide

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 29 September 2015 and 24 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

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)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The sample solutions were taken from the waterbath at various times and the pH of each solution recorded.

Buffers:

See below

Details on test conditions:

The test system used sterile buffer solutions at pH’s 4, 7 and 9.

Performance of the Test
Preparation of the Test Solutions
Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 100 mg/L in the buffer solutions.
The test solutions were split into individual vessels for each data point.
The solutions were shielded from light whilst maintained at the test temperature.

Preliminary Test / Tier 1
Sample solutions at pH 4, 7 and 9 at a nominal concentration of 100 mg/L were maintained at 50.0 ± 0.5 °C for a period of at least 120 hours.

Tier 2
Results from the Preliminary Test / Tier 1 showed it was necessary to undertake further testing at pH 7 and pH 9.

Duration:

120 h

Temp.:

50 °C

Initial conc. measured:

100 mg/L

Number of replicates:

Two

Positive controls:

no

Negative controls:

no

Statistical methods:

No data

Preliminary study:

Preliminary Test / Tier 1
pH 4 at 50.0 ± 0.5 ºC
Less than 10% hydrolysis after 5 days at 50 °C, equivalent to a half-life of greater than 1 year at 25 °C.

pH 7 at 50.0 ± 0.5 ºC
The extent of hydrolysis indicated that further testing (Tier 2) was required to estimate the rate constant and half-life at 25 °C.

pH 9 at 50.0 ± 0.5 ºC
The extent of hydrolysis indicated that further testing (Tier 2) was required to estimate the rate constant and half-life at 25 °C.

Transformation products:

not measured

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.007 h-1

DT50:

99.4 h

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

182 d

Details on results:

Tier 2
pH 7 at 40.0 ± 0.5 ºC
Slope = -1.75E-02
kobs = 4.03E-02 hour-1
t½ = 17.2 hours

pH 7 at 50.0 ± 0.5 ºC
Slope = -3.55E-02
kobs = 8.18E-02 hour-1
t½ = 8.47 hours

pH 7 at 60.0 ± 0.5 ºC
Slope = -0.127
kobs = 0.291 hour-1
t½ = 2.38 hours

pH 9 at 50.0 ± 0.5 ºC
Slope = -2.33E-03
kobs = 5.36E-03 hour-1
t½ = 129 hours

pH 9 at 60.0 ± 0.5 ºC
Slope = -9.03E-03
kobs = 2.08E-02 hour-1
t½ = 33.3 hours

pH 9 at 70.0 ± 0.5 ºC
Slope = -2.73E-02
kobs = 6.29E-02 hour-1
t½ = 11.0 hours

pH 7 Arrhenius Data
From the graph of the data, the rate constant and half-life at 25 °C have been estimated to be as follows:
k = 6.97E-03 hour-1
t½ = 99.4 hours

pH 9 Arrhenius Data
From the graph of the data, the rate constant and half-life at 25 °C have been estimated to be as follows:
k = 1.59E-04 hour-1
t½ =4.36E+03 hours
= 182 days

Identification of Hydrolysis Products – Tier 3
Due to the complex nature of the test item, no attempt was made to elucidate the structure of the hydrolysis product. Furthermore, as the use of ion-pairing reagents is not acceptable in HPLC-MS and the test item is extremely complex, any further analysis would result in the parent test item components and the hydrolysis product essentially co-eluting.
That said, as the test item is a reactive dye, it can be postulated that the hydrolysis products are most likely due to removal of one or more of the reactive groups, possibly the terminal sulphooxy- groups, with the central, dye group remaining essentially unaffected. This would also be the reason why the hydrolyzed solutions retained a similar, orange color compared to the non-hydrolyzed solutions.

Tier 2

The mean peak areas relating to the standard and sample solutions are shown in the following table:

Tier 2

Solution	Mean peak area
Standard 101 mg/L, pH 7	9.7861 x 10^6
Standard 101 mg/L, pH 7	9.7915 x 10^6
Initial Sample A, pH 7, 50 ºC	1.0237 x 10^7
Initial Sample B, pH 7, 50 ºC	1.0175 x 10^7
2 Hour Sample A, pH 7, 50 ºC	7.7457 x 10^6
2 Hour Sample B, pH 7, 50 ºC	7.7066 x 10^6
4 Hour Sample A, pH 7, 50 ºC	5.9876 x 10^6
4 Hour Sample B, pH 7, 50 ºC	5.9388 x 10^6
5.5 Hour Sample A, pH 7, 50 ºC	4.9347 x 10^6
5.5 Hour Sample B, pH 7, 50 ºC	4.9076 x 10^6
Standard 100 mg/L, pH 7	1.2080 x 10^7
Standard 101 mg/L, pH 7	1.2186 x 10^7
24 Hour Sample A, pH 7, 50 ºC	1.4822 x 10^6
24 Hour Sample B, pH 7, 50 ºC	1.4805 x 10^6
26 Hour Sample A, pH 7, 50 ºC	1.3197 x 10^6
26 Hour Sample B, pH 7, 50 ºC	1.3179 x 10^6
28 Hour Sample A, pH 7, 50 ºC	1.1785 x 10^6
28 Hour Sample B, pH 7, 50 ºC	1.1872 x 10^6
Standard 105 mg/L, pH 7	1.0573 x 10^7
Standard 103 mg/L, pH 7	1.0222 x 10^7
Initial Sample A, pH 7, 40 ºC	1.0016 x 10^7
Initial Sample B, pH 7, 40 ºC	1.0004 x 10^7
3 Hour Sample A, pH 7, 40 ºC	8.5062 x 10^6
3 Hour Sample B, pH 7, 40 ºC	8.4418 x 10^6
5 Hour Sample A, pH 7, 40 ºC	7.7111 x 10^6
5 Hour Sample B, pH 7, 40 ºC	7.6385 x 10^6

Solution	Mean peak area
Standard 107 mg/L, pH 7	9.0253 x 10^6
Standard 106 mg/L, pH 7	9.2483 x 10^6
3 Hour Sample A, pH 7, 60 ºC	3.8943 x 10^6
3 Hour Sample B, pH 7, 60 ºC	3.8901 x 10^6
4 Hour Sample A, pH 7, 60 ºC	2.6513 x 10^6
4 Hour Sample B, pH 7, 60 ºC	2.6521 x 10^6
5 Hour Sample A, pH 7, 60 ºC	2.0477 x 10^6
5 Hour Sample B, pH 7, 60 ºC	2.0386 x 10^6
5.5 Hour Sample A, pH 7, 60 ºC	1.7922 x 10^6
5.5 Hour Sample B, pH 7, 60 ºC	1.7857 x 10^6
Standard 107 mg/L, pH 7	1.0194 x 10^7
Standard 106 mg/L, pH 7	1.0274 x 10^7
Initial Sample A, pH 7, 60°C	1.0258 x 10^7
Initial Sample B, pH 7, 60°C	1.0207 x 10^7
1 Hour Sample A, pH 7, 60 ºC	7.2085 x 10^6
1 Hour Sample B, pH 7, 60 ºC	7.0282 x 10^6
2 Hour Sample A, pH 7, 60 ºC	5.2002 x 10^6
2 Hour Sample B, pH 7, 60 ºC	5.1387 x 10^6
Standard 107 mg/L, pH 7	9.1421 x 10^6
Standard 106 mg/L, pH 7	9.1094 x 10^6
24 Hour Sample A, pH 7, 40 ºC	2.9884 x 10^6
24 Hour Sample B, pH 7, 40 ºC	2.9530 x 10^6
27 Hour Sample A, pH 7, 40 ºC	2.6082 x 10^6
27 Hour Sample B, pH 7, 40 ºC	2.5818 x 10^6
29 Hour Sample A, pH 7, 40 ºC	2.4038 x 10^6
29 Hour Sample B, pH 7, 40 ºC	2.3733 x 10^6
Standard 102 mg/L, pH 7	8.4321 x 10^6
Standard 104 mg/L, pH 7	8.4813 x 10^6
48 Hour Sample A, pH 7, 40 ºC	1.2223 x 10^6
48 Hour Sample B, pH 7, 40 ºC	1.2317 x 10^6

Solution	Mean peak area
Standard 102 mg/L, pH 9	1.2443 x 10^7
Standard 102 mg/L, pH 9	1.2471 x 10^7
Initial Sample A, pH 9, 50 ºC	1.2928 x 10^7
Initial Sample B, pH 9, 50 ºC	1.3116 x 10^7
Standard 102 mg/L, pH 9	1.2608 x 10^7
Standard 101 mg/L, pH 9	1.2579 x 10^7
45 Hour Sample A, pH 9, 50 ºC	1.0333 x 10^7
45 Hour Sample B, pH 9, 50 ºC	1.0332 x 10^7
Standard 100 mg/L, pH 9	1.2572 x 10^7
Standard 105 mg/L, pH 9	1.3221 x 10^7
118.5 Hour Sample A, pH 9, 50 ºC	6.9992 x 10^6
118.5 Hour Sample B, pH 9, 50 ºC	6.9138 x 10^6
Standard 102 mg/L, pH 9	1.2723 x 10^7
Standard 101 mg/L, pH 9	1.2602 x 10^7
167 Hour Sample A, pH 9, 50 ºC	5.4056 x 10^6
167 Hour Sample B, pH 9, 50 ºC	5.4571 x 10^6
Standard 100 mg/L, pH 9	1.2621 x 10^7
Standard 103 mg/L, pH 9	1.2824 x 10^7
288 Hour Sample A, pH 9, 50 ºC	2.9182 x 10^6
288 Hour Sample B, pH 9, 50 ºC	2.8804 x 10^6
Standard 110 mg/L, pH 9	1.3863 x 10^7
Standard 104 mg/L, pH 9	1.2999 x 10^7
336 Hour Sample A, pH 9, 50 ºC	2.1118 x 10^6
336 Hour Sample B, pH 9, 50 ºC	2.2353 x 10^6
Standard 106 mg/L, pH 9	1.2127 x 10^7
Standard 101 mg/L, pH 9	1.1564 x 10^7
Initial Sample A, pH 9, 60 /70 ºC	1.1416 x 10^7
Initial Sample B, pH 9, 60 / 70 ºC	1.1715 x 10^7
Standard 105 mg/L, pH 9	1.1916 x 10^7
Standard 102 mg/L, pH 9	1.2047 x 10^7
24 Hour Sample A, pH 9, 60 ºC	8.1738 x 10^6
24 Hour Sample B, pH 9, 60 ºC	8.0143 x 10^6
22 Hour Sample A, pH 9, 70 ºC	3.0048 x 10^6
22 Hour Sample B, pH 9, 70 ºC	3.0003 x 10^6
26 Hour Sample A, pH 9, 70 ºC	2.4392 x 10^6
26 Hour Sample B, pH 9, 70 ºC	2.3609 x 10^6

Solution	Mean peak area
Standard 105 mg/L, pH 9	1.3472 x 10^7
Standard 103 mg/L, pH 9	1.3222 x 10^7
49 Hour Sample A, pH 9, 60 ºC	4.6617 x 10^6
49 Hour Sample B, pH 9, 60 ºC	4.6961 x 10^6
43 Hour Sample A, pH 9, 70 ºC	8.5175 x 10^5
43 Hour Sample B, pH 9, 70 ºC	8.0930 x 10^5
49 Hour Sample A, pH 9, 70 ºC	6.2715 x 10^6
49 Hour Sample B, pH 9, 70 ºC	6.0995 x 10^6

The test item concentrations at the given time points are shown in the following tables:

pH7 at 40.0 ± 0.5 ºC

Time (Hours)	Concentration (g/L)		% of mean initial concentration
	A	B	A	B
0	0.101	0.100	-	-
3	8.54 x 10^-2	8.47 x 10^-2	85.0	84.3
5	7.74 x 10^-2	7.68 x 10^-2	77.0	76.3
24	3.48 x 10^-2	3.44 x 10^-2	34.6	34.2
27	3.04 x 10^-2	3.01 x 10^-2	30.2	29.9
29	2.80 x 10^-2	2.76 x 10^-2	27.9	27.5
48	1.49 x 10^-2	1.50 x 10^-2	14.8	14.9

pH7 at 50.0 ± 0.5 ºC

Time (Hours)	Concentration (g/L)		% of mean initial concentration
	A	B	A	B
0	0.105	0.105	-	-
2	7.97 x 10^-2	7.93 x 10^-2	75.9	75.5
4	6.16 x 10^-2	6.11 x 10^-2	58.7	58.2
5.5	5.08 x 10^-2	5.05 x 10^-2	48.4	48.1
24	1.23 x 10^-2	1.23 x 10^-2	11.7	11.7
26	1.09 x 10^-2	1.09 x 10^-2	10.4	10.4
28	9.75 x 10^-3	9.82 x 10^-3	9.28	9.35

pH7at 60.0 ± 0.5 ºC

Time (Hours)	Concentration (g/L)		% of mean initial concentration
	A	B	A	B
0	0.106	0.106	-	-
1	7.48 x 10^-2	7.30 x 10^-2	70.4	68.7
2	5.40 x 10^-2	5.34 x 10^-2	50.8	50.2
3	4.53 x 10^-2	4.52 x 10^-2	42.6	42.6
4	3.08 x 10^-2	3.08 x 10^-2	29.0	29.0
5	2.38 x 10^-2	2.37 x 10^-2	22.4	22.3
6	2.08 x 10^-2	2.08 x 10^-2	19.6	19.5

pH9at 50.0 ± 0.5 ºC

Time (Hours)	Concentration (g/L)		% of mean initial concentration
	A	B	A	B
0	0.106	0.107	-	-
45	8.34 x 10^-2	8.34 x 10^-2	78.2	78.2
118.5	5.56 x 10^-2	5.50 x 10^-2	52.2	51.5
167	4.33 x 10^-2	4.37 x 10^-2	40.6	41.0
288	2.33 x 10^-2	2.30 x 10^-2	21.9	21.6
336	1.68 x 10^-2	1.78 x 10^-2	15.7	16.7

pH9 at 60.0 ± 0.5 ºC

Time (Hours)	Concentration (g/L)		% of mean initial concentration
	A	B	A	B
0	9.95 x 10^-2	0.102	-	-
24	7.06 x 10^-2	6.92 x 10^-2	70.0	68.7
49	3.63 x 10^-2	3.66 x 10^-2	36.0	36.3

pH9 at 70.0 ± 0.5 ºC

Time (Hours)	Concentration (g/L)		% of mean initial concentration
	A	B	A	B
0	9.95 x 10^-2	0.102	-	-
22	2.60 x 10^-2	2.59 x 10^-2	25.7	25.7
26	2.11 x 10^-2	2.04 x 10^-2	20.9	20.2
43	6.64 x 10^-3	6.31 x 10^-3	6.58	6.26
49	4.89 x 10^-3	4.75 x 10^-3	4.85	4.72

The Arrhenius plots wereconstructed using the data shown in the following tables:

pH 7 Arrhenius Data

T (ºC)	T (K)		kobs(hr^-1)	Ln kobs
40	313.15	3.19 x 10^-3	4.03 x 10^-2	-3.21
50	323.15	3.10 x 10^-3	8.18 x 10^-2	-2.50
60	333.15	3.00 x 10^-3	0.291	-1.23

pH 9 Arrhenius Data

T (ºC)	T (K)		kobs(hr^-1)	Ln kobs
50	323.15	3.10 x 10^-3	5.36 x 10^-3	-5.23
60	333.15	3.00 x 10^-3	2.08 x 10^-2	-3.87
70	343.15	2.91 x 10^-3	6.29 x 10^-2	-2.77

Validation

Linearity

The linearity of the detector response with respect to concentration was assessed for each pH (4, 7 and 9) over the nominal concentration range of 25 to 200mg/L (n =7). The correlation curves were satisfactory with first order correlation coefficients (r) of 1.0000, 0.9999 and 0.9997 being obtained for pH 4, 7 and 9 respectively.

Specificity

Matrix matched blank solutions (relevant buffer solution) were injected with every set of samples and no peak was detected at the sample retention timeof the test itempeak.

Precision

The precision of the analytical method was assessed at a nominal concentration of 100 mg/L (n = 5). The results were satisfactory withrelative standard deviation (RSD)results of 4.53% at pH 4, 1.99% at pH 7 and 1.91% at pH 9.

Repeatability of Injection

The repeatability of injection was assessedusing a standard solutionatanominal concentration of 100mg/L (n = 10). The results were satisfactory with relative standard deviationresultsof ≤1.54%.

 Recovery(Accuracy)

Recovery of analysis of the sample extraction procedure was assessed at a nominal sample concentration of 100 mg/L (n = 5), at each of pH 4, pH 7 and pH 9. The results are presented in the following table:

Sample ID	pH	Nominal concentration (mg/L)	Measured concentration (mg/L)	Recovery (%)	Mean recovery (%)	Standard deviation	RSD (%)
4a	4	106	104	97.8	96.0	4.35	4.53
4b	4	141	141	99.2
4c	4	104	104	100
4d	4	109	101	92.2
4e	4	100	90.5	90.5
7a	7	144	146	101	98.3	1.95	1.99
7b	7	116	113	97.0
7c	7	123	123	99.6
7d	7	114	111	97.0
7e	7	105	102	96.8
9a	9	109	111	101	103	1.97	1.91
9b	9	110	117	106
9c	9	105	108	103
9d	9	101	102	102
9e	9	106	108	102

Validity criteria fulfilled:

yes

Conclusions:

The estimated rate constants and half-lives at 25 °C of the test item are:

At pH 4 the estimated half-life at 25 °C was >1 year
At pH 7 the estimated rate constant at 25 °C was 6.97E-03. The estimated half-life at 25 °C was 99.4 hours.
At pH 9 the estimated rate constant at 25 °C was 1.59E-04. The estimated half-life at 25 °C was 182 days

As the pH 7 and pH 9 results indicate that the rate of hydrolysis increases with a decrease in pH, it can be concluded that the test item will hydrolyse extremely rapidly at pH 4. However, as this wasn’t indicated by the pH 4 results, it is likely that the main “test item” peak in the pH 4 standards and samples was due to a stable, hydrolysis product (as opposed to the parent test item).

Executive summary:

Hydrolysis as a Function of pH. Assessment of hydrolytic stability was carried out using a procedure designed to be compatible with Method C7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004. The estimated rate constants and half-lives at 25 °C are shown in the following table:

pH	Rate constant (hr^-1)	Estimated half-life at 25 °C
4	-	>1 year
7	6.97 x 10^-3	99.4 hours
9	1.59 x 10^-4	182 days

As the pH 7 and pH 9 results indicate that the rate of hydrolysis increases with a decrease in pH, it can be concluded that the test item will hydrolyse extremely rapidly at pH 4. However, as this wasn’t indicated by the pH 4 results, it is likely that the main “test item” peak in the pH 4 standards and samples was due to a stable, hydrolysis product (as opposed to the parent test item).

Description of key information

The estimated rate constants and half-lives at 25 °C of the test item are:

- At pH 4 the estimated half-life at 25 °C was >1 year

- At pH 7 the estimated rate constant at 25 °C was 6.97E-03. The estimated half-life at 25 °C was 99.4 hours.

- At pH 9 the estimated rate constant at 25 °C was 1.59E-04. The estimated half-life at 25 °C was 182 days.

As the pH 7 and pH 9 results indicate that the rate of hydrolysis increases with a decrease in pH, it can be concluded that the test item will hydrolyse extremely rapidly at pH 4. However, as this wasn’t indicated by the pH 4 results, it is likely that the main “test item” peak in the pH 4 standards and samples was due to a stable, hydrolysis product (as opposed to the parent test item)