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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to evaluate the rate and completeness of metabolism of Propylene Glycol Dibenzoate in human and rat primary cryopreserved hepatocytes via measurement of the parent compound, and quantitation of Benzoic acid in the culture media post-incubation.
GLP compliance:
no
Remarks:
This study was performed under non-GLP conditions. Because the study was conducted in vitro, no human or animal subjects were used. All work was performed with appropriate local health regulations and ethical approval.

Test material

Constituent 1
Chemical structure
Reference substance name:
Propane-1,2-diyl dibenzoate
EC Number:
242-894-7
EC Name:
Propane-1,2-diyl dibenzoate
Cas Number:
19224-26-1
Molecular formula:
C17H16O4
IUPAC Name:
2-(benzoyloxy)propyl benzoate
Specific details on test material used for the study:
Propylene glycol dibenzoate; Batch no: PR111910A
Form: Liquid
Radiolabelling:
no

Test animals

Species:
other: human and rat primary cryopreserved hepatocytes

Administration / exposure

Route of administration:
other: In Vitro test
Vehicle:
unchanged (no vehicle)
Details on exposure:
Experimental Procedure: The test article was incubated in duplicate, for each timepoint, with cryopreserved hepatocytes at 37°C. The cells were thawed; viable cells were counted and equilibrated according to the supplier’s directions. After 30 min equilibration at 37°C with gentle agitation, the test article was added to the cell suspension to achieve desired final concentration of 1 µM. The cell suspension was incubated at 37°C. At the indicated times, samples were removed and mixed with an equal volume of ice-cold acetonitrile (containing the IS internal standard propranolol) to stop the reaction and precipitate proteins. Stopped reactions were kept on ice for at least ten minutes. The samples were centrifuged to remove precipitated protein, and supernatants were analyzed by LC/MS/MS to quantify the % parent compound remaining.

Data analysis was done to calculate % parent remaining by assuming zero minute time point peak area ratio (analyte/IS) as 100% and dividing the remaining time point peak area ratios by the zero minute time point peak area ratio value. Data was subjected to a fit first-order decay model to determine half-life. A graph of log (ln) of peak area ratio against time was plotted to calculate the slope. Subsequently, half-life and intrinsic clearance values were calculated using the equations below:

Elimination rate constant (k) = (- slope)
Half-life (t1/2) min = 0.693/k
Intrinsic Clearance (CLint) (ml/min/million cells) = (Vx0.693)/t1/2
V= incubation volume ml/number of cells
Duration and frequency of treatment / exposure:
Human hepatocytes: 0, 15, 30, 60, 120 minutes
Rat hepatocytes: 0, 15, 30, 60, 120 minutes
Doses / concentrations
Dose / conc.:
10 other: µM
No. of animals per sex per dose / concentration:
Cell count: 0.5 x 10^6 viable cells/mL
Details on study design:
Please see 'Any other information on materials and methods incl. tables'

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The parent compound was not detectable at the 15 minute time point, suggesting complete metabolism by that time point. Benzoic acid was detected at 9.7 mM at the 15 minute time point. While complete metabolism of a dibenzoate compound would be expected to yield a 2:1 molar ratio of benzoic acid:parent compound, the fact that only roughly 50% of the expected levels of benzoic acid were detected may be explained by the observation that the remaining benzoic acid is further metabolized after the 15 minute time point. The most likely explanation for this discrepancy is that the initial levels of benzoic acid upon addition of the parent compound were higher, and 50% metabolism of the benzoic acid occurred between T0 and 15 minutes. Propylene Glycol (PG) is the other metabolic by product that was expected but the study authors were unable to develop a LC or GCMS method for derivatized or underivatized PG. Two different derivatization procedures (Boronic Acid and Benzoyl Chloride) were unsuccessful.

Any other information on results incl. tables

Table 2. Data Summary: Hepatocyte Stability

Test Article

Species

Clearance (µl/min/million cells)

Half Life

(mins)

Avg % Remaining at Last Point

Comments

Propylene Glycol Dibenzoate

Humans

NR

NR

0.00%

Highly

metabolized

Rats

NR

NR

0.00%

Highly

metabolized

 

Midazolam

Humans

40.8

33.9

8.39%

Phase I Control

Rats

97.0

14.3

1.15%

 

Verapamil

Humans

46.9

29.6

5.78%

Phase I Control

Rats

65.6

21.1

1.59%

 

7-Hydroxy-4-(trifluoromethyl) coumarin

Humans

107.2

12.9

0.60%

Phase II Control

Rats

109.3

12.7

5.03%

NR: Not reportable due to no measurable compound after T0 time point

Table 3. Quantitation of Benzoic Acid in Human Hepatocytes

Human

Concentration (mM)

Time (minutes)

Replicate 1

Replicate 2

Average

120

1.9

1.65

1.8

60

4.87

4.22

4.5

30

7.52

7.02

7.3

15

11

8.38

9.7

0

BLOQ

BLOQ

NR

 

Table 4. Quantitation of Benzoic Acid in Rat Hepatocytes

Rat

Concentration (mM)

Time (minutes)

Replicate 1

Replicate 2

Average

120

3.76

4.22

4.0

60

4.22

6.01

5.1

30

7.59

6.58

7.1

15

9.48

10

9.7

0

BLOQ

BLOQ

NR

Applicant's summary and conclusion

Conclusions:
PGDB was shown to be rapidly metabolized in both in vitro human and rat hepatocytes. Benzoic acid was formed and appeared to undergo metabolism after the 15-minute timepoint. Due to the unsuccessful analytical development of LC or GCMS method for derivatized or underivatized propylene glycol (Boronic Acid and Benzoyl Chloride derivatization procedures), no propylene glycol could be detected.
Executive summary:

The objective of this study was to evaluate the rate and completeness of metabolism of Propylene Glycol Dibenzoate in human and rat primary cryopreserved hepatocytes via measurement of the parent compound, and quantitation of Benzoic acid in the culture media post-incubation.

 

The test article was incubated in duplicate, for each timepoint, with cryopreserved hepatocytes at 37°C. The cells were thawed; viable cells were counted and equilibrated according to the supplier’s directions. After 30 min equilibration at 37°C with gentle agitation, the test article was added to the cell suspension to achieve desired final concentration of 1 µM. The cell suspension was incubated at 37°C. At the indicated times, samples were removed and mixed with an equal volume of ice-cold acetonitrile (containing the IS internal standard propranolol) to stop the reaction and precipitate proteins. Stopped reactions were kept on ice for at least ten minutes. The samples were centrifuged to remove precipitated protein, and supernatants were analyzed by LC/MS/MS to quantify the % parent compound remaining. 

 

Data analysis was done to calculate % parent remaining by assuming zero minute time point peak area ratio (analyte/IS) as 100% and dividing the remaining time point peak area ratios by the zero minute time point peak area ratio value. Data was subjected to a fit first-order decay model to determine half-life. A graph of log (ln) of peak area ratio against time was plotted to calculate the slope. Subsequently, half-life and intrinsic clearance values were calculated.

 

Propylene Glycol Dibenzoate was rapidly metabolized in both human and rat hepatocytes. The parent compound was not detectable at the 15 minute time point, suggesting complete metabolism by that time point. Based on the rapid nature of the disappearance, a half-life could not be calculated. Benzoic acid was detected at 9.7 mM at the 15 minute time point. While complete metabolism of a dibenzoate compound would be expected to yield a 2:1 molar ratio of benzoic acid:parent compound, the fact that only roughly 50% of the expected levels of benzoic acid were detected may be explained by the observation that the remaining benzoic acid is further metabolized after the 15 minute time point. The most likely explanation for this discrepancy is that the initial levels of benzoic acid upon addition of the parent compound were higher, and 50% metabolism of the benzoic acid occurred between T0 and 15 minutes. Propylene Glycol(PG) was the other metabolic by product that was expected but the laboratory was unable to develop a LC or GCMS method for derivatized or underivatized PG. Two different derivatization procedures (Boronic Acid and Benzoyl Chloride) were unsuccessful.