Cyclohexanol, 4-[[(2-amino-3,5-dibromophenyl)methyl]amino]-, hydrobromide (1:1), trans-

Partition coefficient

Administrative data

Endpoint:

partition coefficient

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-01-21 to 1999-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted under GLP conditions in accordance with the official OECD guideline No. 117 (1989).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted March 30, 1989)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method A.8 (Partition Coefficient)

Version / remarks:

(December 1992)

GLP compliance:

not specified

Type of method:

HPLC method

Partition coefficient type:

octanol-water

Test material

Test material information

Constituent 1

Reference substance name:

Mucosolvan HBr

IUPAC Name:

Mucosolvan HBr

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Mucosolvan HBr
- Physical state: beige solid
- Stability under storage: stable
- Storage condition of test material: at room temperature in the dark

Study design

Analytical method:

high-performance liquid chromatography

Results and discussion

Partition coefficient

Type:

log Pow

Partition coefficient:

2.8

Temp.:

24.5 °C

pH:

8

Details on results:

Determination of pKa values:
Using a computer calculation program, the pKa values from Mucosolvan HBr were calculated to be 7.2 and 0.55 (both basic groups) and 16.1 (acidic group). Based on this, it was concluded that there is not a pH in the range 2-8 (i.e. the pH range in which the HPLC method is applicable) at which the test substance is not ionised. From this, it was decided to perform a first test without using a buffer in the mobile phase. Based on the results of this test, it was decided to perform a second test using a mobile phase buffered at pH=8. It was assumed that the test substance is for >80% in its non-ionised form at this pH.

Main test 1:
In the HPLC chromatograms of Mucosolvan HBr, using detection at 210 nm, one test substance peak was observed. The retention time of this peak was below t0 (the retention time of the unretarded component).
The t0 was determined to be 0.924 minutes as a mean value of both measurements (i.e. 0.923 and 0.925 minutes).
The mean values of the retention times, k´ values, log k´ values, log Pow and Pow values are summarized in Table 1.
The temperature of the mobile phase during the measurements was 25.5+-0.5°C.
To check the number of test substance peaks, the test solution, the corresponding blank and 2,4-DDT were injected in a second HPLC-system (75/25 (v/v) acetonitrile/Milli-Q water. No extra test substance peaks were observed.

Main test 2:
In the HPLC chromatograms of Mucosolvan HBr, using detection at 210 nm, two tests substance peaks were observed.
The t0 was determined to be 0.959 minutes as a mean value of both measurements (i.e. 0.956 and 0.962 minutes).
The mean values of the retention times, k´ values, log k´ values, log Pow and Pow values are summarized in Table 2.
The temperature of the mobile phase during the measurements was 24.5+-0.5°C.
To check the number of test substance peaks, the test solution, the corresponding blank and 2,4-DDT were injected in a second HPLC-system (60/40 (v/v) acetonitrile/0.05M phosphate buffer pH=8.. No extra test substance peaks were observed.

Any other information on results incl. tables

The results of the calculation method and the main tests using the HPLC method were not in agreement. The HPLC method is considered a more accurate method than the calculation method. Using the buffered mobile phase, two test substances peaks were observed in the HPLC chromatogram. It was assumed that the peak with a retention time smaller than t0 derives from ionised test substances whereas the peak at 13.69 minutes derives from non-ionised test substance. Using a non buffered mobile phase, only ionised test substance was observed (retention time below t0). According to the guidelines, the test should be performed with non-ionised test substance. Therefore, the result for the peak at 13.69 minutes from the test using the buffered mobile phase is reported as the partition coefficient (n-octanol/water) of Mucosolvan HBr.

Table 1: Results of main test 1.

Substance	tr*	k´	log k´	log Pow	Pow
Reference substance
Ethylmethylketone	1.65	0.786	-0.104	0.3
Nitrobenzene	5.51	4.965	0.696	1.9
Toluene	12.76	12.81	1.11	2.7
Bromobenzene	15.47	15.74	1.20	3.0
1,4 -dichlorobenzene	24.67	25.70	1.41	3.4
Biphenyl	38.95	41.15	1.61	4.0
Test substance	0.655	n.a.	n.a.	<0.3	<2.0

Table 2: Results of main test 2.

Substance	tr*	k´	log k´	log Pow	Pow
Reference substance
Ethylmethylketone	1.76	0.832	-0.080	0.3
Nitrobenzene	5.95	5.200	0.716	1.9
Toluene	13.55	13.13	1.11	2.7
Bromobenzene	16.33	16.03	1.20	3.0
1,4 -dichlorobenzene	25.88	25.99	1.41	3.4
Biphenyl	40.68	41.42	1.62	4.0
Test substance
peak 1	0.854	n.a.	n.a.	<0.3	<2.0
peak 2	13.69	13.28	1.12	2.8**	657

* Mean value of the retention times of both chromatograms

** Interpolated from the regression line: log k´ = 0.465 log Pow - 0.188 (r=0.998, n=6)

n.a. Not applicable

Applicant's summary and conclusion

Conclusions:

The partition coefficient (n-octanol/water) of Mucosolvan HBr is 657 (log Pow = 2.8) at 24.5 +- 0.5°C.

Executive summary:

The determination of the partition coefficient (n-octanol/water) was based on the following guidelines:

OECD Guidelines for Testing of Chemicals, Guideline No. 117: ”Partition coefficient (n-octanol/water) High Performance Liquid Chromatography (HPLC) method” (adopted March 30, 1989).

EEC directive 92/69 EEC, Part A, Methods for the determination of physic-chemical properties, A.8: “Partition coefficient”, EEC Publication no. L383, December 1992.

Calculation from the structural formula:

From the structural formula of Mucosolvan HBr, the logarithm of the partition coefficient (n-octanol/water), log Pow was calculated to be 3.8 (Pow = 5848), using the Rekker calculation method.

HPLC method:

According to the EEC and OECD guidelines, the partition coefficient (n-octanol/water) has to be determined at a pH at least one unit above the pKa for a basic group and at least one unit below the pKa for an acid group. The pKa values for Mucosolvan HBr were calculated using a computer calculation program to be 7.2 and 0.55 for basic groups and 16.1 for an acidic group in the test substance molecule. Based on this, it was concluded that there is not a pH in the range 2-8 (i.e. the pH range in which the HPLC method is applicable) at which the test substance is not ionized. From this, it was decided to perform a first test without using a buffer in the mobile phase. Based on the results of the test, it was decided to perform a second test using a mobile phase buffered at pH=8. It was assumed that the test substance is for >80% in its non-ionised form at this pH.

Conclusion:

The results of the calculation method and the main tests using the HPLC method were not in agreement. The HPLC method is considered a more accurate method than the calculation method. Using the buffered mobile phase, two test substance peaks were observed in the HPLC chromatogram. It was assumed that the peak with a retention time smaller than t0 derives from ionized test substance whereas the peak at 13.69 minutes derives from non-ionised test substance. Using a non buffered mobile phase, only ionized test substances was observed (retention time below t0). According to the guidelines, the test should be perfomed with non-ionised test substance. Therefore, the result for the peak at 13.69 minutes from the test using the buffered mobile phase is reported as the partition coefficient (n-octanol/water) of Mucosolvan HBr.

The partition coefficient (n-octanol/water) of Mucosolvan HBr is 657 (log Pow = 2.8) at 24.5 +- 0.5°C.