Ethylene carbonate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

2 (reliable with restrictions)

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene carbonate

EC Number:

202-510-0

EC Name:

Ethylene carbonate

Cas Number:

96-49-1

Molecular formula:

C3H4O3

IUPAC Name:

1,3-dioxolan-2-one

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Ethylene carbonate
- Purity: > 99%
- Physical state: fine crystals

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23019647G0
- Expiration date of the lot/batch: October 22, 2016
- Purity test date: no data
- Purity: 99.93 area-% determined by the sponsor (non-GLP).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: storage stability: Room temperature. The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor BASF SE, and the Sponsor holds this responsibility.
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Ethylene carbonate was dissolved in methanol (75%) and further diluted at a concentration of 100 mM (solution for spiking incubates) and 10 mM (stock solutions for preparation of solutions for calibration samples and quality control samples, respectively).
Working solutions for spiking analytical samples: 0.2 - 8 mM in methanol (75%)

Radiolabelling:

no

Test animals

Species:

other: in vitro: the applied matrix was Wistar rat blood

Details on test animals or test system and environmental conditions:

All incubations were conducted in polypropylene cups. For the study described, the applied matrix was Wistar rat blood (with anticoagulant Lithium-heparine).
The incubation solutions (n=3 per test condition) were prepared by adding 2 µL of the 100-fold concentrated working solution of 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. For each time point, individual samples (n=3) were prepared.

After 0, 0.5, 1, 5 and 30 minutes, respectively, the corresponding samples were chemically inactivated by addition of TCA containing protein precipitation reagent (+ internal standard).

Administration / exposure

Vehicle:

other: methanol

Details on exposure:

Control system: 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.
Test system: Blood freshly prepared from Wistar rats (using Lithium Heparine as anticoagulant, stored for < 24h at 4°C before use.)
Test Substance: 1 mM propylene carbonate
Positive Conrol (Reference substance): 1 mM ethylene carbonate
Negative control matrix: blood treated with TCA reagent
Time points: 0, 0.5, 1, 5, 10, 30 min (test system) as well as 0, 30 min (control system) at 37°C

Duration and frequency of treatment / exposure:

no data

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: mM

Remarks:

Test substance propylene carbonate

Doses / concentrations 2

Dose / conc.:

1 other: mM

Remarks:

Reference substance ethylene carbonate

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

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.

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.

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.

Details on dosing and sampling:

Incubations were conducted at 37°C on a horizontal shaker at 650 rpm. For each time point, individual samples (n=3) were prepared.
After 0, 0.5, 1, 5 and 30 minutes, respectively, the corresponding samples were chemically inactivated by addition of TCA containing protein precipitation reagent (+ internal standard). In all samples, the test item (or reference item) and its hydrolysis products) were quantified by LC/MS analysis.

Statistics:

Data analysis:
Mean, SD, slope, R²: Calculation with Microsoft Excel 2007 functions AVERAGE, STDEV, SLOPE and RSQ respectively; %CV: ratio of STDEV/AVERAGE * 100
% remaining: amount of parent compound in the samples expressed in percentage of remaining compound compared to time point zero (= 100%)
% formation: amount of metabolite in the samples expressed in percentage of formation compared to parent amount at time point zero (= 100%)
half-life (t1/2): Estimation using the rate of parent dissappearance using t1/2 = ln2/-k; t1/2= half life (min), k = slope from the linear regression of log (test compound) versus time plot (1/min)
Plot: course of compound amount over time

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

not examined

Details on distribution in tissues:

not examined

Details on excretion:

not examined

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

propylene glycol for the test item propylene carbonate and ethylene glycol for the positive control ethylene carbonate

Any other information on results incl. tables

The positive control item ethylene carbonate was 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).

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).

These findings 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.

For the negative control samples with inactivated blood and the incubations in phosphate buffer half-life values could not be calculated as the compounds were stable within these matrices. Just as well, there was no formation of glycols as hydrolysis products.

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 in blood is fast and occurred with maximum degradation rates of 0.68 μmol/(ml x min) and 0.14 μmol/(ml x min) respectively. 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:

The objective of the study was to assess the in vitro degradation rates of propylene carbonate and ethylene carbonate (positive control) in blood of Wistar rats. Both substances were incubated in Wistar rat blood over a time span of 30 minutes. Concerning ethylene carbonate 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).

This finding supported the assumption that the half-life of ethylene carbonte in blood is indeed smaller than 15 min as observed in the in vivo study conducted by Hanley et al. (1989).