Reaction mass of [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (1S,2R,4aS,6aR,6aR,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylate and [(2S,3R,4S,5S,6R)-6-[[(2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (4aS,6aR,6aS,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate

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 27 november 2020 to 16 december 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

GLP compliance:

yes (incl. QA statement)

Type of method:

flask method

Key result

Water solubility:

>= 676.5 - <= 677.5 g/L

Temp.:

20 °C

pH:

ca. 8

RESULTS
1 Preliminary estimation
A weight of 0.1 g of test item was shaken at room temperature with increasing volumes of water in a stepwise manner.
After each addition of an amount of water, the mixture was shaken for 10 minutes and the dissolution was assessed visually In order to estimate the solubility of the test item. At the request of the sponsor, additional evaluations were performed.
Test temperature: 19.9C

Amount test item weighed (g)	0.101	0.101	0.101	0.101	0.101	0.101	0.101
mL of water for 0.1 g soluble	0.1	0.5	1	2	10	100	> 100
Approximative solubility g/L	> 1,000	1,000 to 200	200 to 100	100 to 50	50 to 10	10 to 1	< 1
Result (Yes or No)	Not soluble	soluble	---	---	---	---	---

Taking the visual observations into account, the preliminary test shows that the solubility of the test item was obtained at 200 g/L.
The solubility estimated of the test item “Reaction mass of Madecassoside and Terminoloside ” in water at ambient temperature was comprised between 1,000 g/L and 200 g/L.

 

2 Analytical validation
2.1 Used analytical equipment

Material (FCBA reference)	Detection (FCBA reference)	Manufacturer
HPLC/UV (HPLC 1003)	PDA 2998 Waters (DETE 1008)	Waters

HPLC conditions for the determination of benfluralin
Data handling system: Empower 3
Columns: Ascentis express C18 (10 cm * 4.6 mm internal diameter – 5 μm)
Pre-column: Security guard 4 x 3 mm
Column temperature: 40°C
Mobile phase composition: Gradient elution

Time (minutes)	%A	%B
0.0	85	15
1	85	15
20	50	50
25	50	50

Mobile phase A: Ammonium phosphate buffer (1.15g/L)
Mobile phase B: Acetonitrile/Methanol (70/30)
Flow rate: 1.4mL/min
Injection volume: 10 μL
Detector: UV set at 204 nm
Retention time:

Approximatively 9.8 minutes for Madecassoside

 

2.2 Preparation of interference solution (specificity)
The aqueous phase was analysed directly by HPLC/UV.

2.3 Preparation of accuracy samples
The matrix distilled water was fortified with the reference item at 2 fortification levels (40 mg/L and 200 mg/L) in order to assess the performance of the method according to the criteria described in the paragraph 9.3. Three independent
samples were prepared for each level of spiking.The spiked samples were analysed directly by HPLC/UV.

2.4 Specificity
Specificity was studied by carrying out once an analysis of the matrix (distilled water). The specificity was assessed by checking for any interference in HPLC/UV at the retention time of Madecassoside (identified with the reference item).
No interference showed at retention time of compound: Madecassoside.
The results obtained for the specificity were compared to the acceptance criteria:

Active substance to quantify	Criteria of acceptance	Conformity
Reaction mass of Madecassoside and Terminoloside”	No interference or blank values (non-fortified) should not exceed 30% of the LOQ. If this exceeded, detailed justification is required.	Yes

On the basis of these results, the applied method to quantify the test item “Reaction mass of Madecassoside and Terminoloside” in the distilled water is considered as specific.

 

2.5 Linearity
Linearity (response function) was examined in order to check the linearity of the response for the four compounds.
Calibration solutions in the range of 28 mg/L to 220 mg/L on seven calibration spots with single determination (28 mg/L - 40 mg/L – 60 mg/L – 100 mg/L – 150 mg/L – 200 mg/L and 220 mg/L (10 % highest point) were prepared within blank matrix samples (evaporated extract). A linear regression and its correlation coefficient were calculated.

Calibration range in mg/L	Regression line - equation (Y = peak area, X = concentration in mg/L)	Regression line – correlation coefficient r
28 to 220 mg/L with single determination at 7 concentrations	Y = 1.781604*103*X – 2.352909*102	0.999533

The calibration was found to be linear, with the correlation coefficient 0.999533, (r > 0.99).
The results obtained for the linearity were compared to the acceptance criteria:

Acceptance criteria	Active substances to quantify	Criteria of acceptance	Conformity
SOP PRQ 2934 [6]	Madecassoside	The equation of the calibration line and the correlation coefficient (r) must be reported and a typical calibration plot submitted. Correlation coefficient r should be > 0.99.	Yes

On the basis of these results, the applied method to quantify “Madecassoside” is considered to be linear on the concentration range 24.5 mg/L to 60.5 mg/L corresponds to 28 mg/L to 220 mg/L in aqueous phase.
On the basis of these results, the applied method to quantify the test item “Reaction mass of Madecassoside and Terminoloside” in the aqueous phase is considered linear on the concentration range 28- 220 mg/L corresponding to 28
mg/L to 220 mg/L in the aqueous phase.

 

2.6 Precision and Accuracy
Precision and accuracy were carried out by one operator and on one analytical serie. The validation conditions were obtained by using the same method and the same equipment.
Precision was expressed by a relative standard deviation of repeatability RSDr (%).
For the levels of concentration, the recovery rate was calculated by comparison between the theoretical "target value" and the average determined value of the 3 samples of spiked ultra-pure water. Accuracy was assessed through recovery
rate calculations.
Precision and recovery rate at 40 mg/L

Sample ID	Concentration in aqueous phase (mg/L)	Recovery rate (%)
20/1171F/1/a/1/a/Dop1 40mg/L	41.08	102.70
20/1171F/1/a/2/a/Dop2 40mg/L	41.06	102.65
20/1171F/1/a/3/a/Dop3 40mg/L	40.73	101.83
Average (%)	---	102.4
Standard deviation (%)	---	0.489
RSDr (%)*	---	0.5

*Relative standard deviation of repeatability calculated from rounded values of the mean and the standard deviation.
The result obtained for the precision was compared to the acceptance criteria according to SOP PRQ 2934 [6]

Active substance to quantify	Criteria of acceptance Validation criteria	Results	Conformity
Madecassoside	The precision of the method must be reported as repeatability of recovery at each fortification level. The RSD should be ≤ 20% per level. In certain justified cases higher variability may be accepted.	RSDr = 0.5 % (n=3)	Yes

The result obtained for the accuracy was compared to the acceptance criteria according to SOP PRQ 2934 [6].

Active substance to quantify	Criteria of acceptance Validation criteria	Results	Conformity
Madecassoside	The accuracy of the method should be reported as mean recovery. Mean recoveries for each level should be in the range 70-110%. Lower recoveries may be acceptable for difficult analytes, providing precision data are acceptable.recoveries may be acceptable for difficult analytes, providing precision data are acceptable.	Mean recovery = 102.4% (n=3) Within 70-110%	Yes

On the basis of these results, the applied method to quantify the Madecassoside is considered precise and accurate at 40 mg/L in the aqueous phase. All acceptance criteria have been satisfied.

Precision and recovery rate at 200 mg/L

Sample ID	Concentration in aqueous phase (mg/L)	Recovery rate (%)
20/1171F/1/a/4/ Dop1 200mg/L	191.92	95.96
20/1171F/1/a/4/ Dop2 200mg/L	193.31	96.65
20/1171F/1/a/4/ Dop3 200mg/L	197.06	98.53
Average (%)	---	97.0
Standard deviation (%)	---	1.330
RSDr (%)*	---	1.4

*Relative standard deviation of repeatability calculated from rounded values of the mean and the standard deviation
The result obtained for the precision was compared to the acceptance criteria according to SOP PRQ 2934 [6].

Active substance to quantify	Criteria of acceptance Validation criteria	Results	Conformity
Madecassoside	The precision of the method must be reported as repeatability of recovery at each fortification level. The RSD should be ≤ 20% per level. In certain justified cases higher variability may be accepted.	RSDr = 1.4 % (n=3)	Yes

The result obtained for the accuracy was compared to the acceptance criteria according to SOP PRQ 2934 [6] .

Active substance to quantify	Criteria of acceptance Validation criteria	Results	Conformity
Madecassoside	The accuracy of the method should be reported as mean recovery. Mean recoveries for each level should be in the range 70-110%. Lower recoveries may be acceptable for difficult analytes, providing precision data are acceptable. recoveries may be acceptable for difficult analytes, providing precision data are acceptable.	Mean recovery = 97.0% (n=5) Within 70-110%	Yes

On the basis of these results, the applied method to quantify the Madecassoside is considered precise and accurate at 55 mg/L in the aqueous phase. All acceptance criteria have been satisfied.

2.7 Validation criteria
Results obtained for the Unknown Standards were compared to the acceptance criteria:

Date of analysis	Sample ID	Nominal value	Measured value (mg/L)	Deviation (%)	Conformity +/- 15% / nominal value
November 30, 2020	20/1171a 100 mg/L 8/271120/6	100 mg/L	99.16	-0.8	Yes
November 30, 2020	20/1171a 100 mg/L 8/271120/6	100 mg/L	99.74	-0.3	Yes

Results obtained for the Quality Control (QC) were compared to the acceptance criteria:

Date of analysis	Sample ID	Nominal value	Measured value (mg/L)	Deviation (%)	Conformity +/- 15% / nominal value
November 30, 2020	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	96.83	- 3.2	Yes
November 30, 2020	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	96.66	- 3.3	Yes

The unknown standards and the Quality controls (QCs) are well within 85 mg/L and 115 mg/L.

2.8 Conclusion
In accordance with results of the analytical method validation, all acceptance criteria were satisfied: the applied method is consistent with the requirements of the validation for the quantitative analysis of Madecassoside in aqueous phase
samples.
The validation results are summarised as follows:

Parameter	”Madecassoside
Calibration range Correlation coefficient (r)	28 mg/L to 220 mg/L > 0.99 or greater
Precision	RSDr = 0.5% at 40 mg/L RSDr = 1.4 % at 200 mg/L
Accuracy	Recovery = 102.4 % at 40 mg/L Recovery = 97.0 % at 200 mg/L

After this validation step, the analytical protocol was revised for the quantitative analysis of Madecassoside in aqueous phase samples.
This analytical method for determination of the test item “Reaction mass of Madecassoside and Terminoloside”in the aqueous solutions was successfully validated and was fit to purpose.
After this validation step, the analytical protocol was revised for the quantitative analysis of the test item Reaction mass of Madecassoside and Terminolosid” in aqueous phase:
- Analytical protocol FCBA Chemistry No.484-e version 2: Method for the chemical analysis of “Reaction de
mass of Madecassoside and Terminoloside” in water samples from OECD 105 testing by HPLC/UV. (annex 1)
This document describes the following information: range, preparation conditions and analytical conditions by HPLC/UV, statistical results (specificity, precision, and accuracy).

Examples:
- Annex 2-1: Calibration curve
- Annex 2-2: Chromatogram of an analytical standard at 100 mg/L
- Annex 2-3: Chromatogram of a water spiked at 40 mg/L
- Annex 2-4: Chromatogram of a water spiked at 200 mg/L

 

3 Main test: Flask method
3.1 Preparation for the saturation
The weight, the used volume and the temperatures are presented in the table below:

	Assay 24 hours	Assay 48 hours	Assay 72 hours	Assay 96 hours	Assay 96 hours
Weight of the test item (g)	5.053	5.040	5.032	5.019	5.020
Volume of water (mL)	5	5	5	5	5
Temperature: start of the testing (°C)	30.0 +/- 1	30.0 +/- 1	30.0 +/- 1	30.0 +/- 1	30.0 +/- 1
Temperature after equilibration time (°C)	20+/- 0.5	20+/- 0.5	20+/- 0.5	20+/- 0.5	20+/- 0.5
Strip pH	8	8	8	8	8

3.2 Formula for the calculations

C = (AR∗Vf∗Vd∗D) / (Vext∗1000)
Where:
C = Concentration of the compound in aqueous phase (g/L)
AR = concentration measured by HPLC/UV (mg/L)
Vff = Final volume (first dilution) (10 mL)
Vif= Initial volume (first dilution) (0.1 mL)
Vfs = Final volume (second dilution) (5 mL)
Vis= Initial volume (second dilution) (0.1 mL)

3.3 Results after equilibration time
Linearity (response function) was examined in order to check the linearity of the response.

Date	Calibration range in mg/L	Regression line - equation (Y = peak area, X = concentration in mg/L)	Regression line – correlation coefficient r
02.DEC.20	40 to 200 mg/L with single determination at 5 concentrations	Y = 1.750344*103*X + 6.653046*102	0.999994
03.DEC.20	40 to 200 mg/L with single determination at 5 concentrations	Y = 1.758130*103*X + 1.48812*103	0.999977
08.DEC.20	40 to 200 mg/L with single determination at 5 concentrations	Y = 1.747340*103*X + 4.343242*102	0.999998
15.DEC.20	40 to 200 mg/L with single determination at 5 concentrations	Y = 1.732227*103*X + 1.689378*103	0.999983
15.DEC.20	40 to 200 mg/L with single determination at 5 concentrations	Y = 1.729108*103*X + 2.777412*102	0.999995

   

3.4 Results
The results of the hydrosolubility determination are indicated in the following tables.

Exposure time (hour)	24	48	72	96	120
Sample ID	20/1171F/1/a/T24h	20/1171F/1/a/T48h	20/1171F/1/a/T72h	20/1171F/1/a/T96h	20/1171F/1/a/T120h
Result (mg/L)	141.33	124.61	112.13	133.84	141.62
First dilution - Initial volume of dilution (mL)	0.1	0.1	0.1	0.1	0.1
First dilution - Final volume of dilution (mL)	10	10	10	10	10
Second dilution - Initial volume of dilution (mL)	0.1	0.1	0.1	0.1	0.1
Second dilution - Final volume of dilution (mL)	5	5	5	5	5
Solubility of the test item (g/L)	707	623	561*	669	708

* According to the Dixon’s statistical test, this value is NOT considered to be an outlier value. See comments below.

A summary is presented in the following table:
The test is considered as satisfactory if the concentrations measured in the last vessels not differ more than 15 %.

	Assay 24 hours	Assay 48 hours	Assay 72 hours
Results (g/L)	707	623	561
Deviation T48hrs/T72Hrs	---	---	- 10%

As required by the OECD 105 guideline, the concentrations measured in the two last vessels differ less than 15 % (+10 %), the test was considered to be satisfactory.
The maximum of difference observed on the three last sample is: 707-561 = 146 g/L.
The mean of the minimum and the maximum values is (707+561)/2 = 634 g/L. The relative difference, corresponding to the ratio of the maximum difference and the mean of the minimum and the maximum values is: (146/634)* 100 = 23.0%.
The concentrations measured in the three vessels differ more than 15 % (23%) and consequently, two additional samples were prepared for 96 hours and 120 hours.

	Assay 24 hours	Assay 48 hours	Assay 72 hours	Assay 96 hours	Assay 120 hours
Results (g/L)	707	623	561*	669	708
Average (g/L)	677	677	677	677	677
Deviation T72hrs/T96Hrs	---	---	---	---	+ 5.8

* According to the Dixon’s statistical test, this value is NOT considered to be an outlier value. In the following calculation, the measured value at 72 hours was not taken into account in the firstly.
As required by the OECD 105 guideline, the concentrations measured in the two last vessels differ less than 15 %
(+5.8 %), the test was considered to be satisfactory.
The maximum of difference observed on the four samples is: 708 -623 = 85 g/L.
The mean of the minimum and the maximum values is (708+623)/2 = 665.5 g/L. The relative difference, corresponding to the ratio of the maximum difference and the mean of the minimum and the maximum values is: (85/665.5)* 100 = 12.8%.
The concentrations measured in the four vessels differ less than 15 % (12.8%) and consequently, the water solubility was determined using the mean of the four samples, the water solubility has been determined to 677 g/L.
If the measured value at 72 hours is taken into account in the calculation, water solubility is as follows

	Assay 24 hours	Assay 48 hours	Assay 72 hours	Assay 96 hours	Assay 120 hours
Results (g/L)	707	623	561*	669	708
Average (g/L)	654	654	654	654	654
Deviation T72hrs/T96Hrs	---	---	---	---	+ 5.8

* According to the Dixon’s statistical test, this value is NOT considered to be an outlier value. In the following calculation, the measured value at 72 hours was not taken into account in the firstly.
As required by the OECD 105 guideline, the concentrations measured in the two last vessels differ less than 15 % (+4.6 %), the test was considered to be satisfactory.
The maximum of difference observed on the five samples is: 708-561 = 147 g/L.
The mean of the minimum and the maximum values is (708+561)/2 = 634.5 g/L. The relative difference, corresponding to the ratio of the maximum difference and the mean of the minimum and the maximum values is: (147/634.5)* 100 = 23.2%.
The concentrations measured in the five vessels differ more than 15 % (23.2%), the obtained deviation was not compliant according to the acceptance criteria and for information, the water solubility was determined using the mean of
the five last samples, the water solubility has been determined to 654 g/L.
The difference observed on both water solubility is 677-654 = 23 g/L which represents a deviation of -3.4 % [(654-677)*100]/677 = - 3.4%. This deviation was considered as low.
Consequently, the water solubility of the test item has been determined to 677 g/L.

 

3.5 Validation criteria
Results obtained for the Unknown Standards were compared to the acceptance criteria:

Date of analysis	Sample ID	Nominal value	Measured value (mg/L)	Deviation (%)	Conformity +/- 15% / nominal value
02.12.20	20/1171a 100 mg/L 8/271120/6	100 mg/L	100.44	+ 0.4	Yes
			100.12	+0.1	Yes
03.12.20	20/1171a 100 mg/L 8/271120/6	100 mg/L	99.73	- 0.3	Yes
			99.11	- 0.9	Yes
08.12.20	20/1171a 100 mg/L 8/271120/6	100 mg/L	99.86	- 0.1	Yes
			100.08	+ 0.1	Yes
15.12.20	20/1171a 100 mg/L 8/271120/6	100 mg/L	98.51	- 1.5	Yes
			97.73	- 2.3	Yes
15.12.20	20/1171a 100 mg/L 8/271120/6	100 mg/L	100.28	+ 0.3	Yes
			100.02	0.0	Yes

Results obtained for the Quality Control (QC) were compared to the acceptance criteria:

Date of analysis	Sample ID	Nominal value	Measured value (mg/L)	Deviation (%)	Conformity +/- 15% / nominal value
02.12.20	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	105.59	+ 5.6	Yes
			105.35	+ 5.4	Yes
03.12.20	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	96.55	- 3.5	Yes
			96.83	- 3.2	Yes
08.12.20	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	96.78	- 3.2	Yes
			97.38	- 2.6	Yes
15.12.20	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	95.93	- 4.1	Yes
			95.59	- 4.4	Yes
15.12.20	20/1171Fa QC 100 mg/L 8/271120/10	100 mg/L	97.80	- 2.2	Yes
			97.85	- 2.2	Yes

The unknown standards and the Quality controls (QCs) are well within 85 mg/L and 115 mg/L

Example:
- Annex 2-7: Chromatograms of test item at 24 hours, 48 hours, 72 hours ,96 hours and 120 hours

3.6 Conclusion
In conclusion, the water solubility of the test item “Reaction mass of Madecassoside and Terminoloside ” was determined to be 677 g/L at 20+ +/- 0.5°C.

Conclusions:

In conclusion, the water solubility of the test item “Reaction mass of Madecassoside and Terminoloside ” was determined to be 677 g/L at 20 ± 0.5°C.

Executive summary:

The aim of this study was to determine experimentally the water solubility on the test item ” Reaction de mass of Madecassoside and Terminoloside “ according to the OECD 105 guideline using the flask method.
The study was carried out in accordance with the study plan No.20/1171F/a dated November 26, 2020.
There were three deviations and no amendment during this study.
The results of this study are summarized below:
Four flasks were used to mix 5 x 5 g of the test item with 5 mL of water. Flasks were placed in a temperature controlled incubator at 30°C (monitored and recorded) and under stirring at 180 rpm. After 24 hours, one flask was removed and placed in another temperature controlled oven at 20 +/- 0.5°C during 24 hours. After sampling in the aqueous phase, the concentration of the test item was then determined following the analytical method HPLC/UV. The other flasks were
treated similarly after initial equilibration at 30°C for 2, 3, 4 and 5 days respectively. The pH of samples was measured at 24 hours, 48 hours, 72 hours 96 hours and 120 hours. The temperature was monitored and recorded during the test and the temperature was well maintained at 20 +/- 0.5°C.

Water solubility results for each replicate are presented below:
- T24 hours: Test item concentration : 707 g/L ; pH strip: 8
- T48 hours: Test item concentration : 623 g/L ; pH strip: 8
- T72 hours: Test item concentration: see comments § 10.3.4 ; pH strip: 8
- T96 hours: Test item concentration: 669 g/L ; pH strip: 8
- T120 hours: Test item concentration: 708g/L ; pH strip: 8
As required by the guideline, the concentration measured in the last vessels do not differ by more than 15 %. Thus, as shown by these results, the water solubility of the test item is 677 g/L.

Conclusion:
In conclusion, the water solubility of the test item “Reaction mass of Madecassoside and Terminoloside ” was determined to be 677 g/L at 20 ± 0.5°C.

Description of key information

In conclusion, the water solubility of the test item “Reaction mass of Madecassoside and Terminoloside ” was determined to be 677 g/L at 20 ± 0.5°C.

Key value for chemical safety assessment

Water solubility:

677 g/L

at the temperature of:

20 °C