[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

Water solubility

Link to relevant study record(s)

Reference

Endpoint:

water solubility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2022-12-02 to 2023-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Deviations:

no

GLP compliance:

yes

Type of method:

flask method

Key result

Water solubility:

>= 15 - <= 660 µg/L

Temp.:

20 °C

pH:

6.99

Check of temperature

Table 1:    Temperature at Pre‑Incubation (30 C) and Thermostatisation Phase (20 C)     (measured hourly)

Intended temperature [°C]	Measured temperature [°C]
	min.	max.	Mean value ± SD
30.0 ± 0.5	30.03	30.09	30.05 ± 0.02
20.0 ± 0.5	19.95	20.05	19.98 ± 0.02

The pH‑values were determined with the remaining test item mixtures in the original test vessels, which were kept constant at 20± 0.5 C.

Test temperature [°C]	Pre‑incubation time [h]	Replicate	Measured pH	Mean value
20	24	1	7.025	6.990
		2	6.994
	48	1	7.085
		2	6.993
	72	1	6.921
		2	6.923
		Control	7.063	-

Check of Tyndall effects

No Tyndall effects were observed for any test item sample.

 

Water Solubility Results

No significant signals were obtained for the control sample

Table 3:    Water Solubility Results at 20 °C for the Dimethyl Ammonium Component

Preincubation time	Replicate No.	Test item [µg/L]
		Concentration^1)	Mean
24 h	1	2.829	11.412
	2	19.994
48 h	1	24.935	45.354
	2	71.772
72 h	1	3.689	13.207
	2	22.756
Mean (24 - 72 h)			24.324
Deviation [%] (24 - 72 h)^2)			324

Table 4:           Water Solubility Results at 20°C for the Benzylsulphonate Component

Preincubation time	Replicate No.	Test item [µg/L]
		Concentration^1)	Mean
24 h	1	162.287	340.529
	2	518.771
48 h	1	561.354	2857.914
	2	5154.475
72 h	1	483.240	760.175
	2	1037.109
Mean (24 - 72 h)			1320
Deviation [%] (24 - 72 h)^2)			739

1) Mean value of two injections, dilution factor of 2 and exact weight of the standard taken into account

2) Calculated according to formula 1

Results : The analysis showed no valid results with highly varying concentrations with no correspondence of the incubation time.

 

Re‑Analysis with Changed Sample Preparation

Preface : The measured concentrations for the two injections of the same sample showed no significant change and thus the samples were only injected once. The following additional changes were made. Please note that the method with these changes were not fully validated

Dilution medium : All samples, including calibration standards, were prepared in pure acetonitrile

Injection volume: 1 µL (instead of 4 µL)

Calibration range:  0.5 – 100 µg/L (instead of 0.1 – 10 µg/L)

Sample preparation : For each replicate sample, a vial with a volume of at least 1.5 mL were filled with 0.75 mL acetonitrile and weighed. Approx. 0.75 mL of the filtrate were directly given into the pre-filled vials. The exact volume of the filtrate was determined by determining of the difference in weight (assuming a density of the filtrate of 1 g/mL), corresponding to a nominal dilution factor of 2. The exact dilution factor was calculated taking the respective volumes into account. This sample was further diluted with dilution factors of 2 (0.4 mL of the sample with 0.4 mL acetonitrile) and 5 (0.2 mL sample and 0.8 mL acetonitrile), respectively, leading to a total of 3 diluted samples with respective dilution factors of 2, 4 and 10 for each of the replicates.

 

Table 5:    Results of the Re‑Analysis for the Dimethyl Ammonium Component

Preincubation time	Replicate No.	Total dilution factor
		2	4	10
		Concentration^1)[µg/L]
24 h	1	14.7	81.7	24.5
	2	90.3	145.2	146.4
48 h	1	118.7	171.9	162.2
	2	298.7	606.5	660.0
72 h	1	27.1	50.4	54.6
	2	97.7	172.3	143.5

1) Dilution factors and exact weight of the standard taken into account

 

Table 6:    Results of the Re‑Analysis for the Benzylsulphonate Component

Preincubation time	Replicate No.	Total dilution factor
		2	4	10
		Concentration^1) [µg/L]
24 h	1	14.7	52.1	20.0
	2	102.6	76.2	105.4
48 h	1	130.8	117.5	122.0
	2	496.9	506.1	489.4
72 h	1	38.8	33.8	30.5
	2	183.9	159.4	135.3

1) Dilution factors and exact weight of the standard taken into account

Results:  The re‑analysis, with minimized effects of the sample preparation, did not improve the homogeneity of the results. Additionally, another issue occurred in that for some samples the results were not homogenous for the different dilution factors (which were taken into account in the tables given above).

Re‑Analysis of Several Aliquots of a Single Replicate (non GLP)

Preface : As a test for the repeatability of the sample preparation, a total of 6 aliquot samples were prepared in the same manner as described in part 5.5 from a single replicate sample of the water solubility test. Replicate 1 of the samples with the pre-incubation time of 48 h was used.

The analysis was performed using only 3 concentrations for calibration (1, 10 and 100 µg/L)

Table 7:    Analysis of six Aliquots for one Replicate for the Dimethyl Ammonium Component

Replicate No.	Dimethyl Ammonium Component	Benzylsulphonate Component
	Concentration^1)[µg/L]
1	118.6	75.0
2	134.5	83.1
1	181.4	125.4
2	172.1	92.7
1	104.2	58.7
2	120.3	67.6
Mean	138.5	83.8
SD	31.3	23.6
RSD [%]	22.6	28.1

1) Exact dilution factors taken into account

Results : The analysis showed that even aliquots of the same sample are varying with a relative standard deviation of approx. 23%, even if the results are in the same range.

Conclusions:

The solubility of test item was determined at 20 +/- 0.5 °C to be in the range of 15 - 660 µg/L (pH 6.990).

Executive summary:

The water solubility of test item was determined according to OECD guideline 105 (1995) and Council Regulation (EC) No. 260/2014, Method A.6.

The solubility of the test item was determined using the flask method at 20 ± 0.5 °C with pre‑incubation at 30 ± 0.5 °C.

The analytical method (LC‑MS/MS, external standard) for the determination of the test item was validated and tested with satisfactory results in regard to linearity, lowest calibration level, limit of quantification, accuracy, precision, stability, specificity, and procedural recovery.

The water solubility of the test item was determined to be in the range of 15 ‑ 660 µg/L (20 °C, pH 6.990).

Centrifugation as a method for the phase separation was excluded by the results of the preliminary investigations.

The analyses of the definitive testing showed that the filtration did not lead to homogenous results, even if no Tyndall effects could be observed.

There is no better method available for the determination of the water solubility. The general solubility of the test item may be estimated within a range with the data obtained.

Description of key information

The substance is insoluble

Key value for chemical safety assessment

Water solubility:

0.66 mg/L

at the temperature of:

20 °C

Additional information

The water solubility has been determined within a range between 15 to 660 µg/L according to the OECD 105. For the chemical safety assessment, the key value of 066 mg/L has been selected to represent the worst case scenario.