Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper (2+) pentasodium 2-[2-{2-amino-5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxyphenyl}diazen-1-yl]-5-({3-carboxy-4-[2-{5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl]phenyl}methyl)benzoate and sodium chloride

Partition coefficient

Administrative data

Endpoint:

partition coefficient

Remarks:

Partition Coefficient (n-octanol/water), HPLC Method / OECD 117 / EU A.24.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 117 (Partition Coefficient (n-octanol / water), HPLC Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU A.24 method

GLP compliance:

no

Remarks:

GLP compliance not required

Type of method:

HPLC method

Partition coefficient type:

octanol-water

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

EC Number:

948-009-7

Molecular formula:

Not applicable for a multi-constituent substance

IUPAC Name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

Test material form:

solid: particulate/powder

Details on test material:

Acid brown 161 batch no. ID151860 CAS no. 85338-16-5
Composition of the muti-constituent substance is included in the report as an extract of the analytical report.

Specific details on test material used for the study:

Acid brown batch no. ID151860; production date: 01. Jan. 2017; expiry date: 01. Oct. 2019. Purity and composition of the substance is included in the report as an extract of analytical report of the substance.

Study design

Analytical method:

high-performance liquid chromatography

Results and discussion

Partition coefficient

Key result

Type:

log Pow

Partition coefficient:

< 0.3

Temp.:

25 °C

pH:

ca. 6

Details on results:

The study was performed using a HPLC with a C18 column. Six reference items with different retention times and thiourea for the determination of the dead time were used to produce a calibration curve, since retention time on hydrophobic columns and POW are correlated.
The reference items were chosen based on the results of the pre-test. One vial was filled with the reference item mix and one vial with the test item solution. The vials were analysed using the HPLC with the program described below. First one injection from the solvent blank methanol/water 65/35 (v/v) was made. Then three injections were measured from the reference item mix, three injections from the test item and again three injections from the reference item mix.
For each reference item, the capacity factor k was calculated from the retention time of thiourea and the retention time of the respective reference item. Please refer to page 4 of the report that includes a summary table.
A calibration function (log k versus log POW, linear fit) was determined using the literature values for POW of the reference items and the retention times in the six determinations.
The chromatogram of the test item gave two peaks. Peak 2 showed increasing areas during the measurements, indicating, that this peak is a result of instability of the test item during measurements. With the calibration function log k versus log Pow, the corresponding log Pow values of the two peaks were determined with.

Peak Mean Area Relative Area Mean Retention log POW
[mAU*min]1 [%] Time [min] ± Standard Deviation
1 2.047 69.40 0.740 *n.c.
2 0.990 30.60 0.970 -1.729 ± 0.094

The dead time of the method was determined as 0.941 ± 0.002 min. Hence, the two peaks lie within the dead time of the method and not within the range of log POWs of the reference items.

Furthermore because peak 2 showed a relative peak area of only 5.1 % for the freshly pre- pared test item solution and is regarded to be a result of a degradation process, this peak was not used for the evaluation of log POW of the test item.

Therefore, the corresponding log POWs should be stated as < 0.3. Thus, the log POW of the test item Acid Brown 161 is < 0.3.

Any other information on results incl. tables

Test item Acid brown 161

Measurement data

The retention times and peak areas of the test item are presented in the following tables

Measurement	RT 1	RT 2
	min.	min.
Measurement 1	0.742	0.972
Measurement 2	0.740	0.972
Measurement 3	0.739	0.967
Mean	0.740	0.970
Standard Deviation	0.002	0.003

Peak Areas Test Items

Measurement	Area Peak 1	RelativeArea Peak1*	Area Peak 2	RelativeArea Peak 2*
	mAU*min	%	mAU*min	%
Measurement 1	2.062	85.33	0.354	14.67
Measurement 2	2.051	67.19	1.002	32.81
Measurement 3	2.027	55.69	1.613	44.31
Mean	2.047	69.40	0.990	30.60
Relative Standard Deviation [%]	0.01	0.22	0.64	0.49

*The areas of peaks 1 and 2 changed in dependence of time: The relative peak area of peak 1 decreased and the relative peak area of peak 2 increased with the time. The test item is regarded to be not stable in the solvent water.

Because peak 2 showed a relative area of only 5.1 % for the freshly prepared test item solution and is regarded to be a result of degradation process, this peak was not used for the final evaluation of log POW of the test item.

Calculated Values

The calculated values are presented in the following table:

Capacity Factor k, log k and log POW Test Item

Peak	Measurement No.	k	log k	log POW
1	1	-0.211	*n.c.	*n.c.
	2	-0.213	*n.c.	*n.c.
	3	-0.215	*n.c.	*n.c.
	Mean			*n.c.
	Standard Deviation			*n.c.
2**	1	0.033	-1.480	-1.675
	2	0.033	-1.480	-1.675
	3	0.028	-1.556	-1.838
	Mean			-1.729
	Standard Deviation			0.094

* n.c. = not calculable, because k is negative; **assumed as degradation product only, therefore not used for the final evaluation of log POW of the test item

log POW was calculated from the capacity factor as follows:

log POW = (log k + 0.6992) / 0.4661

Applicant's summary and conclusion

Conclusions:

Using the correlation log k / log POW, the log POW of the major components of test item Acid Brown 161 was calculated. The test item Acid Brown 161 exhibited two peaks in the chromatogram which both eluted within the dead time of the method.
Peak 2 showed a relative peak area of only 5.1 % for the freshly prepared test item solution and is regarded to be a result of degradation process, because it showed increasing relative peak areas during the measurement sequence. Therefore this peak was not used for the final evaluation of log POW of the test item.
The log POW of peak 1 was not calculable, because the retention time of peak 1 was lower than the retention time of the dead time marker thiourea.

The log POW of the test item is stated as < 0.3.

Executive summary:

The study was performed using a HPLC with a C18 column. Six reference items with different retention times and thiourea for the determination of the dead time were used to produce a calibration curve, since retention time on hydrophobic columns and POW are correlated.

The reference items were chosen based on the results of the pre-test. One vial was filled with the reference item mix and one vial with the test item solution. The vials were analysed using the HPLC with the program described below. First one injection from the solvent blank methanol/water 65/35 (v/v) was made. Then three injections were measured from the reference item mix, three injections from the test item and again three injections from the reference item mix.

For each reference item, the capacity factor k was calculated from the retention time of thiourea and the retention time of the respective reference item.

A calibration function (log k versus log POW, linear fit) was determined using the literature values for POW of the reference items and the retention times in the six determinations.

The chromatogram of the test item gave two peaks. Peak 2 showed increasing areas during the measurements, indicating, that this peak is a result of instability of the test item during measurements. With the calibration function log k versus log Pow, the corresponding log Pow values of the two peaks were determined with:

Peak	MeanArea [mAU*min]1	RelativeArea [%]	Mean Retention Time[min]	log POW ±Standard Deviation
1	2.047	69.40	0.740	*n.c.
2	0.990	30.60	0.970	-1.729±0.094

*n.c.=notcalculable

The dead time of the method was determined as 0.941 ± 0.002 min. Hence, the two peaks lie within the dead time of the method and not within the range of log Pows of the reference items.

Furthermore because peak 2 showed a relative peak area of only 5.1 % for the freshly prepared test item solution and is regarded to be a result of a degradation process, this peak was not used for the evaluation of log Pow of the test item.

Therefore, the corresponding log POWs should be stated as < 0.3. Thus, the log Pow of the test item Acid Brown 161 is < 0.3.