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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:
17 July 2019 to 20 January 2020
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)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method A.8 (Partition Coefficient - HPLC Method)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 830.7570 (Partition Coefficient, n-octanol / H2O, Estimation by Liquid Chromatography)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Type of method:
HPLC method
Partition coefficient type:
octanol-water
Analytical method:
high-performance liquid chromatography
Key result
Type:
log Pow
Partition coefficient:
> 6.5
Temp.:
40 °C
pH:
6.8
Remarks on result:
other: The mean offset adjusted Log Pow for the test substance ranged from < 1.0 (Acetanilide) to > 6.5 (4,4ʹ-DDT).
Details on results:
RESULTS
- A total of eight reference substances were prepared and injected in duplicate (once near the beginning and once near the end of the HPLC sequence). The retention times for one of the reference substances, thiourea, was used to determine the analytical column dead time (t0) for use in calculating capacity factors (k) of the remaining reference substances and the test substance. The mean retention time of thiourea was 0.884 minutes on the UV detector.
- Seven additional reference substances were analysed. The capacity factors of all the reference substances were calculated based upon their retention times on the UV detector.
- The Calculated Log k values for the reference substances are presented in Table 4 (attached).
- Representative reference standard chromatograms are presented in Figure 2 (attached).
- Prior to the initial injection of the reference standards, an injection of a reagent blank solution (75 %:25 % MeOH: H2O, v/v) showed that the reagents used were free of any contaminants, that would impact the final results of the study, and confirmed the peak retention assignments for the reference substances and test substance. Two peaks: one sharp and one board were detected on the CAD in every reference standard and test substance sample injection. These peaks were most likely attributed to instrument and/or solvent contamination since they are present in every standard and sample injection. These peaks did not have any impact on the results of the study since the test substance eluted as multiple peaks with retentions times spanning the entire chromatographic run (see next paragraph). A representative chromatogram of the reagent blank solution is presented in Figure 2 (attached).
- The four 75.0 mg/L test substance solutions were sequentially injected. The test substance eluted as 7 resolved peaks on the CAD. The test substance was not detected on the UV detector. The mean retention times of each CAD peak ranged from a detector offset corrected 0.729 minutes to 19.305 minutes. The capacity factor (k) of each peak was calculated based on the corresponding retention time. Since the first test substance peak eluted at an offset adjusted retention time of 0.799, which was prior to the retention time of the first reference calibration standard, acetanilide, the corresponding log Pow could not be extrapolated. Therefore, the log Pow of the first peak was reported as less than the Log Pow of acetanilide (Log Pow = 1.0). The calculated mean offset adjusted retention time for test substance peaks 1 through 3 are 0.799, 1.113, and 5.897, respectively. The corresponding mean offset adjusted Log Pow for these test substance peaks were < 1.0, 1.69, and 5.35, respectively. Test substance peaks 4, 5, 6 and 7 were forced off the column using a gradient. Therefore, the log Pow for peaks 4, 5, 6 and 7 were extrapolated and are greater than the longest eluting reference standard, 4,4ʹ-DDT (Log POW = 6.5). There was significant variation in the peak abundances in each 75.0 mg/L test substance sample prepared from the same stock solution. The fourth preparation of the test substance resulted in an 8th peak. The additional peak is not reproducible and was not included in the final reporting. The inclusion or exclusion of the 8th peak has no impact on the final result as its Log Pow is greater than the Log Pow of DDT. This variation could be due to interaction of the test substance to the 75:25 MeOH: Ultrapure water (v/v) final solvent composition. Even though the peak abundances varied in each 75.0 mg/L test substance sample, all seven peaks could be clearly seen on the CAD.
- The retention times and n-octanol/water partition coefficients based on CAD data for the test substance are presented in Table 5 (attached).
- A representative chromatogram of 75.0 mg/L test substance solution is presented in Figure 3 (attached).
Conclusions:
Under the chromatographic conditions specified, the test item eluted as seven discrete peaks on the charged aerosol detector (CAD). The corresponding mean offset adjusted n-octanol/water partition coefficients (Log Pow) were a range from less than Acetanilide (i.e., < 1.0) to greater than 4, 4ʹ-DDT (i.e., > 6.5).
Executive summary:

GUIDELINE

The objective of the study was to experimentally estimate the n-octanol/water partition coefficient of the test substance by a reverse-phase high performance liquid chromatography method. The partition coefficient (POW) is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system of two immiscible solvents (n-octanol and water) and serves as an indicator of the bioaccumulation potential of the non-ionised form of a test material in fish. The test was performed based on procedures in the U.S. EPA Product Properties Test Guidelines, OPPTS 830.7570, Partition Coefficient (n-Octanol/Water), Estimation by Liquid Chromatography, OECD Guideline for the Testing of Chemicals, 117, Partition Coefficient (n-octanol/water), High Performance Liquid Chromatography (HPLC) Method, Official Journal of the European Communities No. L383 Method A.8: Partition Coefficient and TSCA Title 40 of the Federal Code of Regulations, Part 796, Section 1570: Partition Coefficient (n-Octanol/Water) – Estimation by Liquid Chromatography.

 

METHODS

Test materials injected into a reversed-phase HPLC mobile phase are retained on the column in proportion to their partitioning behaviour between a hydrocarbon-based stationary phase and an aqueous mobile phase. Hydrophilic compounds are expected to have less interaction with the stationary phase and elute earlier compared with lipophilic compounds. Quantifying this partitioning behaviour is accomplished by calculation of the capacity factor for a given compound injected on column. For a given mobile phase composition and pH, capacity factors were calculated for reference standards of know Log Pow using thiourea to estimate column dead time (i.e., the retention time of an unretained organic compound). The logarithms of the calculated capacity factors were then plotted against published Log Pow values to establish a linear regression equation.

 

Four solutions of test item were prepared in 75 % methanol (MeOH): 25 % ultrapure water (v/v) at a nominal concentration of 75.0 mg/L. The test substance solution was prepared from a freshly prepared stock solution in MeOH. Eight reference standards were prepared in the respective mobile phase at a nominal concentration range (5.00 to 50.0 mg/L) selected to provide desired ultraviolet (UV) detector response. Of these reference substances, thiourea, was used for dead time determination, seven had known Log Pow values given in the OECD 117 guideline and were used as calibration standards. The reference standard preparations were sequentially injected into an HPLC system followed by single injections of each test substance preparation. The calibration reference standards injection sequence was repeated following the test substance injections. The HPLC system was operated under standardised isocratic, reverse-phase operating conditions per the guideline until the retention time corresponding to elution of the last reference material (DDT) was passed. Thereafter (after 15 minutes), a gradient was employed in order to elute the test substance off from the C18-based stationary phase.

 

RESULTS

The four 75.0 mg/L test substance solutions were sequentially injected. The test substance eluted as 7 resolved peaks on the CAD. The test substance was not detected on the UV detector. The mean retention times of each CAD peak ranged from a detector offset corrected 0.729 minutes to 19.305 minutes. The capacity factor (k) of each peak was calculated based on the corresponding retention time. Since the first test substance peak eluted at an offset adjusted retention time of 0.799, which was prior to the retention time of the first reference calibration standard, acetanilide, the corresponding Log Pow could not be extrapolated. Therefore, the Log Pow of the first peak is reported as less than the Log Pow of acetanilide (Log Pow = 1.0). The calculated mean offset adjusted retention time for test substance peaks 1 through 3 are 0.799, 1.113, and 5.897, respectively. The corresponding mean offset adjusted Log Pow for these test substance peaks were <1.0, 1.69, and 5.35, respectively. Test substance peaks 4, 5, 6 and 7 were forced off the column using a gradient. Therefore, the Log Pow for peaks 4, 5, 6 and 7 were extrapolated and are greater than the longest eluting reference standard, 4,4ʹ DDT (Log Pow = 6.5). There was significant variation in the peak abundances in each 75.0 mg/L test substance sample prepared from the same stock solution. The fourth preparation of the test substance resulted in an 8th peak. The additional peak was not reproducible and was not included in the final reporting. The inclusion or exclusion of the 8th peak has no impact on the final result as its Log Pow is greater than the Log Pow of DDT. This variation could be due to interaction of the test substance to the 75:25 MeOH: Ultrapure water (v/v) final solvent composition. Even though the peak abundances varied in each 75.0 mg/L test substance sample, all seven peaks could be clearly seen on the CAD.

 

CONCLUSION

Under the chromatographic conditions specified, the test item eluted as seven discrete peaks on the charged aerosol detector (CAD). The corresponding mean offset adjusted n-octanol/water partition coefficients (Log Pow) were a range from less than Acetanilide (i.e., < 1.0) to greater than 4, 4ʹ-DDT (i.e., > 6.5).

Description of key information

Under the chromatographic conditions specified, the test item eluted as seven discrete peaks on the charged aerosol detector (CAD). The corresponding mean offset adjusted n-octanol/water partition coefficients (Log Pow) were a range from less than Acetanilide (i.e., < 1.0) to greater than 4, 4ʹ-DDT (i.e., > 6.5) (OECD 117, EU Method A.8 and OPPTS 830.7570).

Key value for chemical safety assessment

Log Kow (Log Pow):
6.5
at the temperature of:
40 °C

Additional information

GUIDELINE

The objective of the study was to experimentally estimate the n-octanol/water partition coefficient of the test substance by a reverse-phase high performance liquid chromatography method. The partition coefficient (POW) is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system of two immiscible solvents (n-octanol and water) and serves as an indicator of the bioaccumulation potential of the non-ionised form of a test material in fish. The test was performed based on procedures in the U.S. EPA Product Properties Test Guidelines, OPPTS 830.7570, Partition Coefficient (n-Octanol/Water), Estimation by Liquid Chromatography, OECD Guideline for the Testing of Chemicals, 117, Partition Coefficient (n-octanol/water), High Performance Liquid Chromatography (HPLC) Method, Official Journal of the European Communities No. L383 Method A.8: Partition Coefficient and TSCA Title 40 of the Federal Code of Regulations, Part 796, Section 1570: Partition Coefficient (n-Octanol/Water) – Estimation by Liquid Chromatography.

 

METHODS

Test materials injected into a reversed-phase HPLC mobile phase are retained on the column in proportion to their partitioning behaviour between a hydrocarbon-based stationary phase and an aqueous mobile phase. Hydrophilic compounds are expected to have less interaction with the stationary phase and elute earlier compared with lipophilic compounds. Quantifying this partitioning behaviour is accomplished by calculation of the capacity factor for a given compound injected on column. For a given mobile phase composition and pH, capacity factors were calculated for reference standards of know Log Pow using thiourea to estimate column dead time (i.e., the retention time of an unretained organic compound). The logarithms of the calculated capacity factors were then plotted against published Log Pow values to establish a linear regression equation.

 

Four solutions of test item were prepared in 75 % methanol (MeOH): 25 % ultrapure water (v/v) at a nominal concentration of 75.0 mg/L. The test substance solution was prepared from a freshly prepared stock solution in MeOH. Eight reference standards were prepared in the respective mobile phase at a nominal concentration range (5.00 to 50.0 mg/L) selected to provide desired ultraviolet (UV) detector response. Of these reference substances, thiourea, was used for dead time determination, seven had known Log Pow values given in the OECD 117 guideline and were used as calibration standards. The reference standard preparations were sequentially injected into an HPLC system followed by single injections of each test substance preparation. The calibration reference standards injection sequence was repeated following the test substance injections. The HPLC system was operated under standardised isocratic, reverse-phase operating conditions per the guideline until the retention time corresponding to elution of the last reference material (DDT) was passed. Thereafter (after 15 minutes), a gradient was employed in order to elute the test substance off from the C18-based stationary phase.

 

RESULTS

The four 75.0 mg/L test substance solutions were sequentially injected. The test substance eluted as 7 resolved peaks on the CAD. The test substance was not detected on the UV detector. The mean retention times of each CAD peak ranged from a detector offset corrected 0.729 minutes to 19.305 minutes. The capacity factor (k) of each peak was calculated based on the corresponding retention time. Since the first test substance peak eluted at an offset adjusted retention time of 0.799, which was prior to the retention time of the first reference calibration standard, acetanilide, the corresponding Log Pow could not be extrapolated. Therefore, the Log Pow of the first peak is reported as less than the Log Pow of acetanilide (Log Pow = 1.0). The calculated mean offset adjusted retention time for test substance peaks 1 through 3 are 0.799, 1.113, and 5.897, respectively. The corresponding mean offset adjusted Log Pow for these test substance peaks were <1.0, 1.69, and 5.35, respectively. Test substance peaks 4, 5, 6 and 7 were forced off the column using a gradient. Therefore, the Log Pow for peaks 4, 5, 6 and 7 were extrapolated and are greater than the longest eluting reference standard, 4,4ʹ DDT (Log Pow = 6.5). There was significant variation in the peak abundances in each 75.0 mg/L test substance sample prepared from the same stock solution. The fourth preparation of the test substance resulted in an 8th peak. The additional peak was not reproducible and was not included in the final reporting. The inclusion or exclusion of the 8th peak has no impact on the final result as its Log Pow is greater than the Log Pow of DDT. This variation could be due to interaction of the test substance to the 75:25 MeOH: Ultrapure water (v/v) final solvent composition. Even though the peak abundances varied in each 75.0 mg/L test substance sample, all seven peaks could be clearly seen on the CAD.

 

CONCLUSION

Under the chromatographic conditions specified, the test item eluted as seven discrete peaks on the charged aerosol detector (CAD). The corresponding mean offset adjusted n-octanol/water partition coefficients (Log Pow) were a range from less than Acetanilide (i.e., < 1.0) to greater than 4, 4ʹ-DDT (i.e., > 6.5).