Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-05 to 2009-06-26

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

The test solutions were prepared by twofold dilution of the saturated stock solutions with the corresponding buffer solutions.
F 213 Red concentrations of the solutions were as follows:
In pH 4 buffer solution →1.28 mg/l, (≈7.2*10-4 mM),
in pH 7 buffer solution→1.85 mg/l, (≈1.0*10-3 mM),
in pH 9 buffer solution→2.15 mg/l, (≈1.2*10-3mM),
in pH 9 buffer solution→1.28 mg/l, (≈7.2*10-4 mM) for the hydrolysis at 55 C and at 67°C .
Additionally, control buffer samples were stored at each temperature.
The solutions were stored in screw-cap glass tubes with PTFE septa. Volume of the tubes was 15 ml.
From pH 7 and pH 9 buffer solutions 30 such tubes were placed in the thermostate; from pH 4 solution 6 tubes were stored.
Three tubes were analysed for each temperature in time intervals which provided a minimum of six spaced data points between 10 % and 90 %
hydrolysis of the F 213 Red.
Three samples were analysed from three separate tubes at all analytical occasion of hydrolysis test. Sample volume was 0.8 ml, and this was diluted to 1ml with dilution mixture. One sample was taken from the control vessel and it was directly injected. The pH of each buffer solution was checked with a calibrated pH meter.

Buffers:

Buffer Solution: 5 mM Hexane-1-sulfonic acid sodium salt; 15 mM Potassium dihydrogen phosphate, pH=3 with Phosphoric acid 85 %

Estimation method (if used):

not applicable

Details on test conditions:

The hydrolysis was examined in different buffer solutions.
Temperature: 50 °C ± 0.5 °C, 55 °C ± 0.5 °C, 67 °C ± 0.5 °C.
Light and oxygen: The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects. Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.
All glassware, water and buffer solution were sterilised.

Duration of test

Duration of test 1

Duration:

120 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

1.28 mg/L

Duration of test 2

Duration:

120 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

1.85 mg/L

Duration of test 3

Duration:

72 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

2.15 mg/L

Duration of test 4

Duration:

24 h

pH:

9

Temp.:

55 °C

Initial conc. measured:

1.28 mg/L

Duration of test 5

Duration:

10 h

pH:

9

Temp.:

67 °C

Initial conc. measured:

1.28 mg/L

Number of replicates:

Three replicates.

Positive controls:

no

Negative controls:

yes

Remarks:

Control buffers assessed at each pH both after 2.4 hours and 5 days

Statistical methods:

The chromatograms were evaluated with the help of “LaChrom Chromatogram Processor".
Calculations were carried out using “EXCEL for Windows". The calibration curves were constructed with “STATISTICA for Windows" using weighted linear regression. The factor was 1/concentration.
For pseudo-first order the rate constant (kobs) was determined from the plots of the natural logarithm (ln) of the concentration versus time: (eq. 2 and 3).
lntkCCt*0= (eq. 1)
ln Ct = ln C0 – k *t (eq. 2)
kobs = –slope (eq. 3)
obskt2ln21= (eq. 4)
where t time
t 1/2 half-life period
C0 start concentration
Ct concentration at time point “t”
Applying the Arrhenius equation the rate constant and half-life were calculated at 25°C from the plot of ln k versus 1/T:
ln k = (-E/R*T) + lnA
where E activation energy [kJ/mol]
T absolute temperature [K]
R gas constant [8.314 J/mol*K]
A A-factor, empirical constant

Results and discussion

Preliminary study:

The content of F 213 Red in the different pH buffer samples was determined at the beginning, after 2.4 hours and the end of the test periods.
Three samples were analysed from three separate tubes at the start of hydrolysis test, after 2.4 hours and after 120 hours. Sample volume of 1 ml was diluted twofold with dilution mixture at the start of study and after 2.4 hours. On the end of preliminary test the sample volume of 1.6 ml was diluted to 2 ml. If the preliminary test indicates that 50% or more of the test substance has been hydrolysed in 2.4 hours at 50 °C, or less than 10 % has been hydrolysed after 5 days at each of the three pH values (4, 7 and 9), no further testing is necessary.
The concentration of F 213 Red decreased at pH 4 by 18 % and 62 % , at pH 7 by 23 % and 76 % , and at pH 9 by 19 % and more than 90 % after 2.4 hours resp. after 5 days at 50°C. Based on the results obtained, test substance F 213 Red can be considered as hydrolytically unstable at pH 4, pH 7 and pH 9. Thus, additionally a main study was performed at each of the three pH values.

Test performance:

not applicable

Transformation products:

not measured

Total recovery of test substance (in %)

Total recovery of test substance (in %) 1

% Recovery:

98

pH:

4

Temp.:

50 °C

Duration:

120 h

Total recovery of test substance (in %) 2

% Recovery:

94

pH:

7

Temp.:

50 °C

Duration:

120 h

Total recovery of test substance (in %) 3

% Recovery:

14

pH:

9

Temp.:

50 °C

Duration:

72 h

Total recovery of test substance (in %) 4

% Recovery:

33

pH:

9

Temp.:

55 °C

Duration:

24 h

Total recovery of test substance (in %) 5

% Recovery:

26

pH:

9

Temp.:

67 °C

Duration:

10 h

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.03 h-1

DT50:

25 h

Type:

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

Dissipation DT50 of parent compound 2

pH:

9

Temp.:

55 °C

Hydrolysis rate constant:

0.05 h-1

DT50:

15 h

Type:

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

Dissipation DT50 of parent compound 3

pH:

9

Temp.:

67 °C

Hydrolysis rate constant:

0.13 h-1

DT50:

5 h

Type:

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

Dissipation DT50 of parent compound 4

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 h-1

DT50:

14 d

Type:

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

Other kinetic parameters:

not applicable

Details on results:

not applicable

Any other information on results incl. tables

Measured data at a temperature of 50 °C at the pH4

pH	Sampling time. hour	Concentration of F 213 Red, mg/l		Rate. %	Measured pH
		Test vessels	Mean with the 95% confidence intervals	Hydrolysis %
4	Start	1.28	1.28±0.02	-	4.02
		1.28
		1.30
		Control buffer			4.02
	120	1.27	1.26±0.03	2	4.02
		1.26			4.02
		1.25			4.02
		Control buffer			4.00

  Measured data at a temperature of 50 °C at the pH 7

pH		Sampling time. hour	F 213 Red concentration, mg/l				Hydrolysis %	Measured pH
			Test vessels		Mean with the 95% confidence intervals
7	Start			1.82		1.85±0.07	-	6.99
				1.84
				1.88
				Control buffer				6.98
	72			1.80		1.80±0.06	2	7.00
				1.78				7.00
				1.83				7.01
				Control buffer				7.00
	120			1.69		1.74±0.10	6	7.02
				1.77				7.02
				1.76				7.02
				Control buffer				7.01

Measured data at a temperature of 50 °C at pH 9

Temperature °C	Time hours	F 213 Red concentration, mg/l		Hydrolysis %	Measured pH
		Test vessels	Mean with the 95% confidence intervals
50	Start	2.16	2.15±0.06	-	9.01
		2.17
		2.12
		Control buffer			9.00
	4	2.01	2.04±0.07	5	9.00
		2.06			9.01
		2.06			9.01
		Control buffer			9.01
	6	1.95	1.90±0.10	12	9.02
		1.87			9.02
		1.88			9.02
		Control buffer			9.01
	8	1.79	1.86±0.17	14	9.02
		1.91			9.02
		1.88			9.02
		Control buffer			9.02
	11	1.60	1.65±0.11	23	9.01
		1.69			9.02
		1.66			9.01
		Control buffer			9.01
	13	1.55	1.58±0.07	27	9.02
		1.58			9.02
		1.60			9.01
		Control buffer			9.01
	15	1.49	1.47±0.07	32	9.01
		1.44			9.01
		1.46			9.01
		Control buffer			9.01

Temperature °C	Time hours	F 213 Red concentration, mg/l		Hydrolysis %	Measured pH
		In the Samples	Meanwith the 95% confidence intervals
50	24	1.12	1.15±0.07	46	9.02
		1.16			9.02
		1.18			9.02
		Control buffer			9.01
	29	1.01	1.02±0.05	53	9.02
		1.01			9.01
		1.04			9.02
		Control buffer			9.02
	34	0,84	0.88±0.09	59	9.02
		0.89			9.02
		0.91			9.02
		Control buffer			9.01
	72	0.30	0.30±0.02	86	9.02
		0.31			9.02
		0.31			9.01
		Control buffer			9.01

Measured data atpH 9at 55± 0.5 °C

Temperature °C	Time hours	F 213 Red, mg/l		Hydrolysis %	Measured pH
		In the Samples	Mean with the 95% confidence intervals
55	Start	1.34	1.28±0.15	-	9.02
		1.26
		1.23
		Control buffer			9.02
	1	1.20	1.17±0.06	8	9.03
		1.18			9.04
		1.15			9.02
		Control buffer			9.04
	2	1.13	1.16±0.08	9	9.04
		1.19			9.01
		1.15			9.03
		Control buffer			9.04
	3	1.11	1.08±0.06	15	9.02
		1.08			9.04
		1.06			9.02
		Control buffer			9.03
	5	1.04	1.04±0.02	18	9.03
		1.06			9.03
		1.04			9.04
		Control buffer			9.04
	7	0.96	0.92±0.08	28	9.05
		0.92			9.02
		0.89			9.04
		Control buffer			9.04
	8	0.89	0.85±0.14	34	9.03
		0.86			9.02
		0.78			9.04
		Control buffer			9.01

Temperature °C	Time hours	F 213 Red, mg/l		Hydrolysis %	Measured pH
		In the Samples	Mean with the 95% confidence intervals
55	9	0.84	0.84±0.02	35	9.01
		0.83			9.02
		0.83			9.03
		Control buffer			9.04
	10	0.80	0.78±0.05	39	9.04
		0.76			9.03
		0.79			9.01
		Control buffer			9.03
	12	0.75	0.74±0.06	42	9.02
		0.71			9.02
		0.75			9.03
		Control buffer			9.03
	24	0.41	0.42±0.02	67	9.02
		0.43			9.04
		0.41			9.05
		Control buffer			9.01

Measured data atpH 9at67±0.5 °C

Temperature °C	Time hours	F 213 Red, mg/l		Hydrolysis %	Measured pH
		In the Samples	Mean with the 95% confidence intervals
67	Start	1.34	1.28±0.15	-	9.02
		1.26
		1.23
		Control buffer			9.02
	1	1.22	1.18±0.11	8	9.03
		1.17			9.03
		1.13			9.02
		Control buffer			9.04
	2	1.13	1.09±0.13	14	9.05
		1.06			9.02
		-			9.03
		Control buffer			9.04
	3	0.97	1.00±0.09	22	9.01
		1.04			9.03
		0.98			9.02
		Control buffer			9.03
	4	0.89	0.88±0.03	31	9.03
		0.87			9.02
		0.89			9.03
		Control buffer			9.03
	5	0.74	0.74±0.01	42	9.04
		0.73			9.03
		0.74			9.04
		Control buffer			9.02
	6	0.63	0.64±0.01	50	9.03
		0.64			9.02
		0.64			9.03
		Control buffer			9.04

Temperature °C	Time hours	F 213 Red, mg/l		Hydrolysis %	Measured pH
		In the Samples	Mean with the 95% confidence intervals
67	7	0.53	0.55±0.05	57	9.02
		0.57			9.02
		0.54			9.05
		Control buffer			9.03
	8	0.45	0.47±0.03	63	9.01
		0.48			9.03
		0.47			9.04
		Control buffer			9.04
	9	0.43	0.43±0.01	66	9.04
		0.43			9.03
		0.43			9.03
		Control buffer			9.04
	10	0.33	0.34±0.12	74	9.02
		0.39			9.02
		0.29			9.03
		Control buffer			9.03

Rate constants and half-lives calculated at 25°C

Temperature, °C	pH	kobs	t1/2
25	9	0.002	14 days(344 hours)

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

pH 7 at 25 °C, t 1/2: > 1 year
pH4 at 25 °C, t 1/2: > 1 year
pH9 at 25 °C, t 1/2: 14 days (344 hours)
ph 9 at 50 °C, t 1/2: 25 hours

Executive summary:

The purpose of this study was to evaluate the hydrolysis of F 213 Red in different pH buffer solutions.

The hydrolysis of F 213 Red was observed less than 10 per cent after 5 days (which means T1/2 > 1 year at 25°C) in buffer solutions pH 4 and pH 7, therefore the test item is considered to be hydrolytically stable under the conditions investigated.

The calculated half lives of the hydrolysis reactions at pH 9 are 14 days at 25 °C and 25 hours at 50 °C.