Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Hydrolysis study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The hydrolysis rate of propylene carbonate (test item) and the ethylene carbonate (positive control) was investigated.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
BASF test substance No.: 14/0549-1
Batch identification / No: Lot 43181168E0
CAS No.: 108-32-7
Purity: 99.91%
The characterization of the test substance was performed by the Sponsor and the Sponsor holds this responsibility.
Expiry date: October 22, 2016
Storage stability: Room temperature. The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor and the Sponsor holds this responsibility.
Radiolabelling:
no

Test animals

Species:
other: in vitro
Strain:
Wistar
Details on species / strain selection:
Test system: Blood freshly prepared from Wistar rats (using Lithium Heparine as anticoagulant, stored for < 24h at 4°C before use.)

Administration / exposure

Vehicle:
unchanged (no vehicle)
Details on exposure:
Control system: Incubations in potassium phosphate buffer
Test Substance: 1 mM propylene carbonate
Positive Control (Reference substance): 1 mM ethylene carbonate
Negative control matrix: blood treated with TCA reagent
Duration and frequency of treatment / exposure:
Time points: 0, 0.5, 1, 5, 10, 30 min (test system) as well as 0, 30 min (control system) at 37°C
Doses / concentrations
Remarks:
Doses / Concentrations:
1 mM propylene carbonate (test substance) and ethylene carbonate (reference substance)
Positive control:
Negative controls
For the matrix blood, inactivated controls (=negative controls) were included to demonstrate that hydrolysis of the test item was due to specific in vitro metabolism instead of general chemical instability. In the respective negative controls, test item concentration was 1 mM (n=3). The reference item ethylene carbonate was tested at 1 mM (n=3). Negative control samples were prepared using inactive matrix. For blood, the negative controls were inactivated by addition of TCA containing precipitation reagent before the addition of test item to the incubate. After 0 and 30 minutes, respectively, the corresponding samples
were processed for LC-MS analysis.

Buffer control
In addition, potassium phosphate buffer was used as control matrix to exclude non-metabolic degradation processes; i.e. the finding that the concentrations remained stable over the investigated time suggests that the decrease of the parent compound wase mainly due to specific/enzymatic in vitro metabolism. Potassium buffer samples were generated by spiking 198 μl potassium phosphate buffer (100 mM, pH 7.4) with 2 μl test item/reference item working solutions. The resulting start concentrations were 1 mM. After 0 and 30 minutes, the samples were processed for LC-MS analysis.
Details on study design:
The Wistar rat blood applied in the in vitro experiments reported herein is intended to represent the central compartiment of an organism which contains significant amounts of metabolizing enzymes, i.e. hydrolases. These are proposed to metabolize ester compounds such as the test item propylene carbonate and the reference ethylene carbonate.
Propylene carbonate and the reference ethylene carbonate were tested at a concentration of 1 mM in blood. Sampling time points were selected in order to allow the calculation of an in vitro half-life value, i.e. 0 min, 0.5 min, 1 min, 5 min and 30 min. The incubation solutions (n=3 per test condition) were prepared by adding 2 μl of the 100-fold concentrated working solution to 198 μl of pre-incubated assay matrix (15 min preincubation at 37 °C) resulting in an incubation concentration of 1 mM (test and reference items) with a final solvent concentration of 0.75% solvent. Incubations were conducted at 37°C on a horizontal shaker at 650 rpm. After the respective incubation time the corresponding samples were chemically inactivated by addition of TCA containing protein precipitation reagent (+ internal standard).
The precipitation reagent consisted of water supplemented with trichloro acetic acid and internal standard. (The precipitation reagent for PG/EG with 400 μM ISTD PC-d6/EC-d4 was prepared as follows: TCA (8%) in water: e.g. 20 ml water + 1600 mg trichloro acetic acid (TCA) + 800 μl internal standard stock solution (10 mM). The precipitation reagent for blank samples was TCA (8%) in water: 10 ml water + 800 mg trichloro acetic acid (TCA)). The complete in vitro sample volume (200 μl) containing the test item /reference item after incubation with blood or phosphate buffer were mixed with 200 μl precipitation reagent supplemented with ISTD, vortexmixed for 10 second. After a centrifugation step (e.g. 4000 rpm in centrifuge Eppendorf 5417C or equivalent, for 10 minutes) the supernatant was transferred to a 96 well plate for LC-MS analysis.

In a second step the amount of hydrolysis products propylene glycol and ethylene glycol, respectively, were quantified.

Details on dosing and sampling:
Preparation of calibration standards and quality control samples for glycol analysis
Stock solutions of PG and EG are prepared in water (each 15 mM). Separate stocks are prepared for calibrators and QCs, respectively. The working solutions for spiking the matrix were prepared in water at the concentrations 15 mM, 10 mM, 8 mM, 5.33 mM, 4 mM, 2.67 mM, 1.33 mM (for calibrators) and 8mM, 5.33 and 4mM (for quality control samples). The resulting nominal concentrations of the calibrators were 1800, 1200, 960, 640, 480, 320 and 160 nM, the nominal concentrations of the QC samples were 960, 640 and 480 nM. For the preparation of the sample 88 μl matrix was precipitated with 100 μl precipitation reagent with ISTD and immediately vortexed for 10 seconds. Afterwards 12 μl of the corresponding working solution was added and the samples briefly shaken. After a centrifugation step (e.g. 4000 rpm in centrifuge Eppendorf 5417C or equivalent, for 10 minutes) the supernatant was transferred to a 96 well plate for LC-MS analysis. (The precipitation reagent for PG/EG with 400 μM ISTD PC-d6/EC-d4 was prepared as follows: TCA (8%) in water: e.g. 20 ml water + 1600 mg trichloro acetic acid (TCA) + 800 μl internal standard stock solution (10 mM). The precipitation reagent for blank samples was TCA (8%) in water: 10 ml water + 800 mg trichloro acetic acid (TCA)).

LC-MS for glycol analysis (for both PG and EG)
The analysis was performed under isocratic HPLC conditions using 10 % buffer 1 and 90 % buffer 2. Chromatographic run time is 4 min per injection.
The HPLC column was a Luna (Phenomenex), 150 x 4.6 mm, 3 μm. The HPLC flow rate was 900 μl/min. The column is operated at room temperature. Buffer 1 (organic) was acetonitrile, buffer 2 (aqueous) was water + NaCl (10 mg/l). Autosampler wash solvent 1 was Ethanol /Water (5+95, v/v), autosampler wash solvent 2 was Isopropanol / Water (1+1, v/v). The sample injection volume was 20 μl.

In addition, negative control incubates were performed to exclude non-metabolic degradation processes; i.e. the finding that the concentrations remained stable over the investigated time suggests that the decrease of the parent compound is mainly due to hydrolytically active ingredients of blood. Negative controls were blood samples that were chemically inactivated blood samples (using trichloroacetic acid containing reagent) before
the incubation with the test item propylene carbonate and the reference item ethylene carbonate. Potassium phosphate buffer served as additional control matrix to show that any hydrolysis observed with the biorelevant test matrix blood is due to metabolic hydrolytic activity.

A positive control known for hydrolysis in Wistar rat blood was included in the assay set up in order to demonstrate the suitability of the test systems. As positive control for blood incubations ethylene carbonate was tested at 1 mM, respectively.
Statistics:
-The statistical parameters comprised:
Mean: Calculated with MS Excel 2007, function MITTELWERT
SD: Calculated with MS Excel 2007, function STANDARDABWEICHUNG
The numbers displayed in the result tables are rounded.
%CV: Mean of SD x 100 / Mean (coefficient of variation)
Intraday precision: %CV calculated from the 4-fold samples of each QC level

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
propylene glycol

Applicant's summary and conclusion

Conclusions:
The objective of the study was to assess the in vitro degradation rates of propylene carbonate in blood of Wistar rats and phosphate buffer as negative control matrix. As a positive control compound/reference ethylene carbonate was incubated in parallel in these matrices. From the results of the in vitro incubations it can be concluded that PC (and EC) hydrolysis inblood is fast and occurred with maximum degradation rates of 0.68 μmol/(ml x min) and 0.14 μmol/(ml x min). Thus under the incubation conditions used, nearly complete hydrolysis and stoichiometric formation of propylene glycol (and ethylene glycol) was observed after 5 min and 30 minutes, respectively.
Executive summary:

Propylene carbonate was incubated in Wistar rat blood over a time span of 30 minutes. There was 5.5 % of the start concentration remaining after 5 minutes of incubation. After 30 minutes 2 out of 3 samples showed quantification results below the limit of quantification. The hydrolysis product propylene glycol was formed simultaneously from the reference item at concentrations that corresponded to its turnover/hydrolysis.

The calculated half-life value for propylene carbonate was 0.734 minutes. This corresponds to a turnover of 0.68 μmol/(ml x min). The positive control item ethylene carbonate was also incubated in Wistar rat blood over a time span of 30 minutes. 35.5 % of the start concentration remained after 5 minutes of incubation. After 30 minutes 15.5% of the start concentration was observed. The hydrolysis product ethylene glycol was formed simultaneously from the reference item at concentrations that corresponded to its turnover/hydrolysis. The calculated half-life value for ethylene carbonate was 3.533 minutes. This corresponds to a turnover of 0.14 μmol/(ml x min).

These finding indicate that the hydrolysis occurs faster with the propylene carbonate as compared to the positive control. For both compounds the formation of the corresponding glycols was observed simultaneously.