bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

Water solubility

Link to relevant study record(s)

Reference

Endpoint:

water solubility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-24 to 2019-05-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

Version / remarks:

adopted 72 July, 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Version / remarks:

19 March 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Version / remarks:

March 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI- National Institute of Pharmacy and Nutrition (22.05.2019)

Type of method:

column elution method

Water solubility:

< 2 µg/L

Conc. based on:

test mat.

Temp.:

20 °C

pH:

>= 6.2 - <= 6.97

Details on results:

All chromatographic data were collected and analysed using Analyst 1.6.2 software.
The calibration curves were constructed on the basis of the peak area of the test item. The slope, y-intercept and correlation coefficient were calculated automatically by the computing software. The equation of the calibration curve was then used to calculate the back-calculated concentrations for each calibrator, as well as the concentration of the test item in the test solutions from their measured peak area.
The calculations were performed using Microsoft Office Excel software. Accuracy values were rounded to one decimal place.
The mean value from five consecutive samples taken from the saturation plateau was calculated for each run. The results were given in μg/L.
The measured concentration of the test item in the water solubility samples was below the limit of quantification, therefore the calculations of the standard deviation of the five consecutive samples taken from the saturation plateau, as well as the difference between the means obtained from tests at two flow rates were not applicable.

System suitability

System suitability testing is carried out to determine if the chromatographic system and detection are suitable for the analysis. System suitability was carried out before all analytical RUNs on the specific LC-MS/MS system used for analysis. On each occasion, system suitability consisted of 5 injections of the highest calibration solution followed by a blank (ultrapure water:acetonitrile:tetrahydrofuran=50:49.5:0.5 mixture) and the lowest calibration solution. Measured values were evaluated for retention time, carry-over and system sensitivity.

Acceptance criteria for the highest calibration solution: The peak of the analyte should be discrete and sharp and the CV% for peak area of the injections should be less than 10%. Retention time should be in the acceptance range (4.4 ± 0.2 min).

Acceptance criteria for the blank sample (carry-over): no peak with an area higher than 1/5 than that of the analyte at the lowest calibration solution.

Acceptance criteria for the lowest calibration concentration: Signal/noise ratio should be higher than 10.

During the study, 4 system suitability tests were performed. The overall results are the following:

- The coefficient of variation (CV%) of the peak area ratios of the 5 injection of the highest calibration solution was not more than 2.99%.

- Carry-over was not observed.

- The system sensitivity was adequate for the detection of the LOQ on each occasion.

- The retention times of test item were within the acceptance range.

The system was considered to have performed well during the study.

Selectivity

The specificity of the method was examined by analysis of blank solvent (ultrapure water:acetonitrile:tetrahydrofuran=50:49.5:0.5).

Acceptance criteria: Interference in blank sample must be not more than 20% of the LOQ

No interfering peak was detected at the retention time of the test item.

Linearity

The relationship between the detector response and the concentration was examined. The calibration curve was constructed by using 7 independent standard solutions at 7 levels (1, 2, 5, 10, 20, 50, 100 μg/L). The series were measured on four different days with three replicate injections. The deviation of the back-calculated values from the nominal concentration were determined, accuracy was evaluated.

The accuracy was calculated as follows:

Accuracy [%] =(measured conc.-nominal conc.)/nominal conc. *100

Acceptance criteria: deviation from the nominal concentrations (accuracy) is less than 15% (at the LOQ < 20%) and the correlation coefficient (r) of the calibration curve is not less than 0.99.

The means of the peak areas versus the concentration showed a good linearity in the range of 1 – 100 μg/L. The calibration graph was calculated applying linear regression using the method of least-squares.

The deviation of the back calculated value from the nominal concentration (accuracy) was ≤ ±6.0%, at the LOQ it was ≤ ±4.0% in case of all series. The correlation coefficient (r) was ≥ 0.9995, thus the calibrations fulfil the acceptance criteria.

Measured Water solubility

Measured concentrations of the test item in the samples using two different flow rates are presented in the following tables.

Table: Measured concentration of test item and pH in test samples at 25 mL/hour flow rate

Analytical sampling/time period	Concentration [µg/L]	pH	Analytical sampling/time period	Concentration [µg/L]	pH
Column 1			Column 2
19 hours	0	6.96	61 hours	0	6.72
20 hours		6.73	62 hours		6.43
21 hours		6.40	63 hours		6.35
22 hours		6.20	64 hours		6.35
23 hours		6.20	65 hours		6.30
Mean	0	-	Mean	0	-
CV%	-	-	CV%	-	-
Mean all	0 µg/L

Table: Measured concentration of test item and pH in test samples at 12.5 mL/hour flow rate

Analytical sampling/time period	Concentration [µg/L]	pH	Analytical sampling/time period	Concentration [µg/L]	pH
Column 1			Column 2
43 hours	0	6.96	13 hours	0	6.97
44 hours		6.89	14 hours		6.93
45 hours		6.86	15 hours		6.88
46 hours		6.86	16 hours		6.85
47 hours		6.72	17 hours		6.82
Mean	0	-	Mean	0	-
CV%	-	-	CV%	-	-
Mean all	0 µg/L

No colloidal matter was observed in the fractions eluted from the micro-column. Due to the very low solubility of the test item in water and to prevent its adsorption to vessels, the fractions were twice diluted by an organic solvent mixture (acetonitrile:tetrahydrofuran =99:1). Since the measured concentrations of sample in the water solubility samples were below the quantification limit (1 μg/L, equal to 2 μg/L for water), the concentrations of sample were accepted as zero. The measured peak areas of the test item in the samples were approximately 10% of the peak area of the lowest calibrator.

Conclusions:

Since the measured concentration of the sample in the water solubility samples were below the limit of quantification (1 μg/L, equal to 2 μg/L for water), the concentrations of the test item were accepted as zero.

Executive summary:

The water solubility of the test item was determined at 20 ± 0.5 °C applying the column elution method, following internationally accepted guidelines (OECD 105, EU Method A.6, OPPTS 830.7840) and according to GLP. The analytical method used for the quantification of test item concentration in water solubility samples was based on HPLC, comprising an Inertsil ODS-4 column, mobile phases containing water, acetonitrile and acetic acid, MS/MS detection working at the 557.3→166.2 m/z MRM transition for the sample. Selectivity of the method was acceptable as no significant interfering peak was observed at the retention time of the test item in the blank sample (ultrapure water:acetonitrile:tetrahydrofuran=50:49.5:0.5 mixture). Linearity was acceptable over the range of 1-100 μg/L. The accuracy values of the calibrators were acceptable. The measured concentration of the test item in the water solubility samples were below the limit of quantification (1 μg/L, equal to 2 μg/L for water). Consequently, the water solubility was determined to be below 2 μg/L at 20 °C ± 0.5 °C and pH 6.20-6.97, using the column elution method. This evaluation of the test item concentration in water was done with a state-of-the-art LC/MS-MS-method. Due to the very low water solubility of the test item, it was not possible to determine the accurate water solubility value with this methods.

Description of key information

The water solubility of the test item was determined at 20 ± 0.5 °C applying the column elution method, following internationally accepted guidelines (OECD 105, EU Method A.6, OPPTS 830.7840) and according to GLP. The analytical method used for the quantification of test item concentration in water solubility samples was based on HPLC, comprising an Inertsil ODS-4 column, mobile phases containing water, acetonitrile and acetic acid, MS/MS detection working at the 557.3→166.2 m/z MRM transition for the sample. Selectivity of the method was acceptable as no significant interfering peak was observed at the retention time of the test item in the blank sample (ultrapure water:acetonitrile:tetrahydrofuran=50:49.5:0.5 mixture). Linearity was acceptable over the range of 1-100 μg/L. The accuracy values of the calibrators were acceptable. The measured concentration of the test item in the water solubility samples were below the limit of quantification (1 μg/L, equal to 2 μg/L for water). Consequently, the water solubility was determined to be below 2 μg/L at 20 °C ± 0.5 °C and pH 6.20-6.97, using the column elution method. This evaluation of the test item concentration in water was done with a state-of-the-art LC/MS-MS-method. Due to the very low water solubility of the test item, it was not possible to determine the accurate water solubility value with this methods.

Key value for chemical safety assessment

Water solubility:

2 µg/L

at the temperature of:

20 °C

Additional information

The water solubility was determined to be below 2 μg/L at 20 °C ± 0.5 °C and pH 6.20-6.97, using the column elution method. Value stated as key value for CSA is 2 µg/L as this is the limit of quantification.