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Partition coefficient

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Reference
Endpoint:
partition coefficient
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2016 to 07 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 117 (Partition Coefficient (n-octanol / water), HPLC Method)
Deviations:
yes
Remarks:
The results from the two tests do not agree within ± 0.1 log units as specified in the OECD 117 guideline; however it is felt that these results are acceptable based on the chromatography obtained and the nature of the test material.
GLP compliance:
yes (incl. QA statement)
Type of method:
HPLC method
Partition coefficient type:
octanol-water
Analytical method:
high-performance liquid chromatography
Key result
Type:
log Pow
Partition coefficient:
1.15
Temp.:
25 °C
pH:
7
Key result
Type:
log Pow
Partition coefficient:
1.99
Temp.:
25 °C
pH:
7
Key result
Type:
log Pow
Partition coefficient:
2.36
Temp.:
25 °C
pH:
7
Key result
Type:
log Pow
Partition coefficient:
4.85
Remarks on result:
other: The acid: KOWWIN model
Key result
Type:
log Pow
Partition coefficient:
5.167
Remarks on result:
other: The monoester: KOWWIN model
Key result
Type:
log Pow
Partition coefficient:
10.936
Remarks on result:
other: The diester: KOWWIN model
Details on results:
Distribution Coefficient:
- The test material gave three peaks. The mean log Pow values from the two runs were 1.15, 1.99 and 2.36.
- The results from the two tests do not agree within ± 0.1 log units as specified in the OECD 117 guideline; however it is felt that these results are acceptable based on the chromatography obtained and the nature of the test material.
- Calculated Values: Calculated values for partition coefficient were estimated using the KOWWIN model:
The acid: Hydrophobic, predicted partition coefficient (log Kow) 4.8504.
The monoester: Hydrophobic, predicted partition coefficient (log Kow) 5.1671.
The diester: Partition coefficient (Kow) is difficult to measure experimentally, predicted partition coefficient (log Kow) 10.9361.

Analytical Procedure

- Due to the issues with the validation for the analytical method for water solubility it was decided, in consultation with the Sponsor, that the partition coefficient would only be done at one pH level. As the pH level selected for the water solubility test was pH 7 it was decided that this pH would also be used for the partition coefficient test. Based on this, method validation was only carried out at pH 7.

- Four validation batches were analysed as follows:

Log P Val 1: Method validation for pH 7 buffer saturated with n-octanol.

Log P Val 2: Method validation for n-octanol saturated with pH 7 buffer.

Log P Val 3: Method validation for pH 7 buffer saturated with n-octanol and n-octanol saturated with pH 7 buffer using re-dilutions of samples used for the previous two validation batches.

Log P Val 4: Method validation for pH 7 buffer saturated with n-octanol and n-octanol saturated with pH 7 buffer using freshly prepared samples.

- A successful validation could not be achieved with percent recovery and percent relative standard deviations (%RSD) falling outside the acceptance criteria at different points in each batch. As a result, the analytical method was not used and the partition coefficient was carried out using the OECD guideline 117 HPLC method which method that does not require a validated method.

- A summary of the validation results is given below but because the validation was not successful, full results of each validation batch have not been reported.

- The method was assessed against the criteria detailed in the following sections:

Specificity:

Diluting solvent and blank control solutions were generally free of any components that interfered with the analysis of the test material. However, there were a few instances where the responses were > 30 % of the responses for the limit of quantification samples as follows:

First validation (Log P Val 1) for the  pH 7 buffer saturated with n-octanol control sample had a response of 47 % of the lowest LOQ sample response for the diester.

Second validation (Log P Val 3) for the pH 7 buffer saturated with n-octanol control sample had a response of 40 % of the lowest LOQ sample response for the diester.

Third Validation (Log P Val 4) for pH 7 buffer saturated with n-octanol control sample had a response of 40 % of the lowest LOQ sample response for the acid.

Based on this it cannot be claimed that the analytical method is totally specific for the test material. However, as the analysis was done using a LC-MS/MS TOF instrument with specific ions being monitored it is more likely that the peaks in the controls were due to carryover of the test material rather than interferences from other substances.

Sensitivity:

The lowest calibration standard was 0.001 µg/mL (0.00037 µg/mL as the acid, 0.000190 µg/mL as the monoester and 0.000439 µg/mL as the diester which demonstrated sufficient sensitivity.

The limit of quantification (LOQ) was defined as the lowest validation level that gave acceptable mean recoveries and percent relative standard deviation (%RSD).

Matrix

LOQ for the acid (µg/mL)

LOQ for the monoester (µg/mL)

LOQ for the diester (µg/mL)

pH 7 buffer saturated with n-octanol

Could not be defined as none of the validation batches passed for this constituent

0.190

0.439

n-octanol saturated with pH 7 buffer

979.81
(mean concentration of 5 samples)

501.79
(mean concentration of 5 samples)

1121.56*
(mean concentration of 5 samples)

Linearity:

Response of the LC-MS/MS TOF system to the test material was linear with a 1/X weighting, over the range 0.001 to 0.1 μg/mL (0.000371 to 0.0371 µg/mL as the acid, 0.000190 to 0.0190 µg/mL as the monoester and 0.000439 to 0.0439 µg/mL as the diester, with correlation coefficients of > 0.99. However it should be noted that for three of the validation batches (Log P Val 1, Log P Val 2 and Log P Val 3)  the calibration failed for the diester (correlation coefficients of 0.91076, 0.90273 and 0.96639, respectively).

Accuracy (Recovery):

The accuracy mean recovery values varied between batches with no one batch showing acceptable mean recoveries at all validation levels for all three constituents.

The summary of the acceptability of the accuracy mean recovery results is as follows:

Matrix

Validation batch

Constituent

Acceptable mean recoveries

Low level

Mid-level

High level

pH 7 buffer saturated with
n-octanol

Log P Val 1

Acid

No

No

No

Monoester

No

No

Yes

Diester

No*

No*

No*

Log P Val 3

Acid

No

No

No

Monoester

Yes

No

Yes

Diester

No*

No*

Yes*

Log P Val 4

Aid

No

Yes

No

Monoester

Yes

Yes

No

Diester

Yes

Yes

Yes

n-Octanol saturated with pH 7 buffer

Log P Val 2

Acid

Yes

No

No

Monoester

Yes

No

No

Diester

Yes*

Yes*

Yes*

Log P Val 3

Acid

Yes

No

Yes

Monoester

No

No

No

Diester

No*

No*

No*

Log P Val 4

Acid

Yes

Yes

Yes

Monoester

No

No

Yes

Diester

Yes

Yes

Yes

 

Precision (Repeatability):

The precision %RSD values varied between batches with no one batch showing acceptable recoveries at all validation levels for all three constituents.

The summary of the acceptability of the precision %RSD results is as follows:

 

Matrix

Validation batch

Constituent

Acceptable %RSD

Low level

Mid-level

High level

pH 7 buffer saturated with
n-octanol

 

 

 

 

 

Log P Val 1

Acid

Yes

Yes

Yes

Monoester

Yes

Yes

Yes

Diester

No*

No*

No*

Log P Val 3

Acid

Yes

Yes

Yes

Monoester

Yes

Yes

Yes

Diester

No*

No*

Yes*

Log P Val 4

Acid

No

No

No

Monoester

Yes

No

No

Diester

Yes

Yes

Yes

n-Octanol saturated with pH 7 buffer

Log P Val 2

Acid

Yes

Yes

Yes

Monoester

Yes

Yes

Yes

Diester

No*

Yes*

No*

Log P Val 3

Acid

Yes

Yes

Yes

Monoester

Yes

Yes

Yes

Diester

Yes*

Yes*

Yes*

Log P Val 4

Acid

Yes

Yes

Yes

Monoester

Yes

Yes

Yes

Diester

Yes

Yes

Yes

*The calibration lines for the diester failed (correlation coefficients < 0.99) for batches Log P Val 1, Log P Val 2 and Log P Val 3.

 

- Due to the poor validation results for this method it was considered not possible to successfully validate the analytical method for partition coefficient. This meant that the shake flask and slow stir methods for partition coefficient could not be used. As a consequence so, the OECD 117 HPLC method was used instead.

Interpreting Results

The KOWWIN model uses a "fragment constant" method to predict the partition coefficient (Kow usually reported as the logarithm log Kow). In the "fragment constant" method, a molecule is divided into fragments (atoms or larger functional groups) and the assigned coefficient values for each fragment are added to give the Kow estimate, which is reported as log Kow.

The predicted partition coefficients are interpreted as follows:

 

For log Kow< 1 – the substance is classified as highly soluble in water (hydrophilic).

For log Kow> 4 – the substance is classified as not very soluble in water (hydrophobic).

For log Kow> 8 – the substance is classified as not readily bioavailable.

For log Kow> 10 – the substance is classified as not bioavailable – partitioning properties are difficult to measure experimentally.

The EPI suite has no statistical test that can be used to judge the reliability of the predicted values. The KOWWIN model is a fragment approach so a surrogate method to judge the reliability of the predictions is to check that all the substructures in the compound of interest are represented in the calculations. If all the substructures are in the model domain, and are accounted for, then this increases the confidence in the predictions and the model result may be assumed to provide relevant information.

Method

Stock solutions were prepared by weighing out the standards and dissolving in methanol as follows:

Reference Substance

Weight of Reference Substance Used (mg)

Final Volume of Solution in Methanol (mL)

Final Concentration, Corrected for Purity (µg/mL)

Formamide

212.78

10

21129.05

Acetanilide

41.45

10

4136.71

Benzonitrile

70.21

10

7018.19

Methyl benzoate

53.88

10

5382.61

Ethyl benzoate

59.57

10

5951.04

Bromobenzene

69.63

10

6949.07

Naphthalene

17.61

10

1759.24

Phenanthrene

13.90

10

1363.59

 

These solutions were then diluted further to give working solutions:

Reference Substance

Concentration of Stock Solution (µg/mL)

Volume of Stock Solution Used (mL)

Final Volume in Mobile Phase Diluent (mL)

Final Concentration, Corrected for Purity (µg/mL)

Formamide

21129.05

0.10

10

211.79

Acetanilide

4136.71

0.05

10

20.68

Benzonitrile

7018.19

0.05

10

35.09

Methyl benzoate

5382.61

0.05

10

26.91

Ethyl benzoate

5951.04

0.10

10

59.51

Bromobenzene

6949.07

0.05

10

34.75

Naphthalene

1759.24

0.10

10

17.59

Phenanthrene

1363.59

0.25

10

34.09

 

The amounts of the reference substance and test substance injected on the HPLC-UV system were as follows:

 

Reference Substance

Concentration of Working Solution used (µg/mL)

Volume of Solution Injected (µL)

Amount of Reference Substance Injected (µg)

Formamide

211.79

10

2.11

Acetanilide

20.68

10

0.21

Benzonitrile

35.09

10

0.35

Methyl benzoate

26.91

10

0.27

Ethyl benzoate

59.51

10

0.60

Bromobenzene

34.75

10

0.35

Naphthalene

17.59

10

0.18

Phenanthrene

34.09

10

0.34

Test material

394.20

10

3.942

 

Conclusions:
Under the conditions of this study, the test material gave three peaks. The mean log Pow values from the two runs were 1.15, 1.99 and 2.36.
Calculated values for partition coefficient were estimated using the KOWWIN model:
The acid: Hydrophobic, predicted partition coefficient (log Kow) 4.8504.
The monoester: Hydrophobic, predicted partition coefficient (log Kow) 5.1671.
The diester: Partition coefficient (Kow) is difficult to measure experimentally, predicted partition coefficient (log Kow) 10.9361.
Executive summary:

The partition coefficient of the test material was investigated in accordance with the standardised guideline OECD 117, under GLP conditions.

Due to issues with getting acceptable validation results for the analytical procedure, in aqueous pH buffers saturated with n-octanol and n-octanol saturated with aqueous pH 7 buffers, a suitable validated analytical method could not be obtained. As a result, the partition coefficient could not be carried out using the shake flask method so testing was done following the HPLC method as specified in OECD guideline 117. Testing was done at pH 7 only.

Under the conditions of this study, the test material gave three peaks. The mean log Pow values from the two runs were 1.15, 1.99 and 2.36. The results from the two tests do not agree within ± 0.1 log units as specified in the OECD 117 guideline; however it is felt that these results are acceptable based on the chromatography obtained and the nature of the test material.

Calculated values for partition coefficient were estimated using the KOWWIN model:

The acid: Hydrophobic, predicted partition coefficient (log Kow) 4.8504.

The monoester: Hydrophobic, predicted partition coefficient (log Kow) 5.1671.

The diester: Partition coefficient (Kow) is difficult to measure experimentally, predicted partition coefficient (log Kow) 10.9361.

Description of key information

Under the conditions of this study, the test material gave three peaks. The mean log Pow values from the two runs were 1.15, 1.99 and 2.36.

For the chemical safety assessment, a weighted average log Pow of 1.84 (at 25 °C) was derived, based on the constituents’ experimentally derived log Pow values and their proportion (%) in the substance (see table below).

Constituent

Percentage in substance

Log Pow

Log Pow component

The acid

37.1

1.15

0.43

The monoester

19

1.99

0.38

The diester

43.9

2.36

1.04

Weighted average Log Pow Component

 

1.84

 

Key value for chemical safety assessment

Log Kow (Log Pow):
1.84
at the temperature of:
25 °C

Additional information

The partition coefficient of the test material was investigated in accordance with the standardised guideline OECD 117, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Due to issues with getting acceptable validation results for the analytical procedure, in aqueous pH buffers saturated with n-octanol and n-octanol saturated with aqueous pH 7 buffers, a suitable validated analytical method could not be obtained. As a result, the partition coefficient could not be carried out using the shake flask method so testing was done following the HPLC method as specified in OECD guideline 117. Testing was done at pH 7 only.

Under the conditions of this study, the test material gave three peaks. The mean log Pow values from the two runs were 1.15, 1.99 and 2.36. The results from the two tests do not agree within ± 0.1 log units as specified in the OECD 117 guideline; however it is felt that these results are acceptable based on the chromatography obtained and the nature of the test material.

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