Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jul - 13 Sep 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report which meets basic scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Objective of study:
other: hydrolysis in rat plasma
Principles of method if other than guideline:
Study investigating the in vitro hydrolysis rate of the test substance in rat plasma under physiological conditions
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-(1-methylethoxy) ethyl acetate
- Physical state: clear colourless liquid
- Analytical purity: 99.90% (w/w) ester content
- Lot/batch No.: 57033
- Expiration date of the lot/batch: 2014-11-25
- Storage condition of test material: room temperature in the dark
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: RCC HAN WISTAR
Sex:
not specified
Details on test animals and environmental conditions:
TEST SYSTEM
- Description of the test system: rat (RCC HAN WISTAR) plasma in lithium heparin (Harlan Laboratories, Belton, UK) incubated at 37 ± 0.5 °C.

Administration / exposure

Route of administration:
other: the test substance was dissolved in rat plasma and homogenised
Vehicle:
unchanged (no vehicle)
Details on exposure:
PRINCIPLE OF THE TEST
The test item was prepared in rat plasma and incubated in the dark in a water bath thermostatically controlled at the test temperature of 37 ± 0.5 °C. The concentration of the test substance was determined as a function of time, using a suitable analytical method (spectrometry or GC-MS analysis).

PERFORMANCE OF THE TEST
SCREENING TEST
A screening test was performed to obtain an estimate of the rate of hydrolysis of the test substance. Therefore, an aliquot (0.0149 g) of the test substance was dissolved in 10 mL rat plasma and shaken to homogenise. The sample was scanned on a Perkin Elmer Spectrum Two Infrared spectrometer using an Attenuated Total Reflectance (ATR) assessory over the wavenumber range of 4000 to 650 cm-1. Sample scans were performed against a background scan of both air and rat plasma.
The sample was scanned against background scans of air and rat plasma after intervals of 1, 2 and 4 h.

DEFINITIVE TESTS
PREPARATION OF TEST SOLUTIONS
For each definitive test, stock solutions were produced by dissolving aliquots of the test item in rat plasma to give a nominal concentration of 1.4 g/L (0.01 M) (see Table 1 under “Any other information on materials and methods incl. tables”). For each test, the stock solution was split into separate glass vessels and sealed.

INCUBATION OF TEST SOLUTIONS
The vessels containing the stock solutions were incubated at 37 ± 0.5 °C. The concentration of the test item in the test solutions was analysed initially and after various incubation times. For each time point, a separate vessel was used for analysis. During test 1, the vessels were allowed to cool to approx. room temperature prior to analysis; for tests 2 to 4, the samples were analysed immediately.
Duration and frequency of treatment / exposure:
Duration of incubation of the test substance in rat plasma:
Test 1: 0, 1, 2, 3, 4, 24 and 28 h
Test 2: 0, 15, 30, 45 and 60 min
Test 3: 0, 8, 16, 24, 32, 40 and 48 min
Test 4: 0, 8, 16, 24, 32, 40 and 48 min
Doses / concentrations
Remarks:
Doses / Concentrations:
1.4 g/L (0.01 M)
No. of animals per sex per dose:
For each test and the respective time points, a separate vessel containing the test item dissolved in rat plasma was used for analysis.
Control animals:
other: for GC/MS analysis: matrix blank sample (acetone:rat plasma (9:1 (v/v), filtered) and duplicate standard solutions in acetone:rat plasma (9:1 (v/v), filtered prior to prep. of standards)
Details on dosing and sampling:
ANALYSIS OF PARENT SUBSTANCE IN RAT PLASMA
- Principle: the concentrations in the test solutions for each test and the respective time points were analysed using gas chromatography (GC).
- Calibration standard: duplicate standard solutions were prepared at a nominal concentration of 140 mg/mL in acetone:rat plasma (9:1 (v/v), filtered prior to prep. of standards).
Matrix blank: acetone:rat plasma (9:1 (v/v), filtered)
- Sample preparation: an aliquot of each sample vessel removed from incubation was diluted by a factor of 10 with acetone and filtered.
- GC system and conditions:
GC system: Agilent Technologies 6890
Column: DB-5 (30 m x 0.25 mm id x 0.25 µm film)
Injector mode: Split
Split ratio: 5:1
Injection temperature: 275 °C
Oven temperature programme: initial 40 °C for 2 min; rate 20 °C/min; final 280 °C for 5 min
Flow rate: 1.5 mL/min (constant flow)
Detector type: Flame ionisation detector (FID)
FID detector temperature: 275 °C
Injection volume: 1 µL
Retention time of the parent substance: approx. 4 min

ANALYSIS AND IDENTIFICATION OF HYDROLYSIS PRODUCTS IN RAT PLASMA
- Principle: a standard solution, matrix blank as well as initial and 48 min samples from definitive test 3 were analysed using GC-MS (mass spectrometry).
GC system: Agilent Technologies 6890
Column: DB-5 (30 m x 0.25 mm id x 0.25 µm film)
Injector mode: Split
Split ratio: 5:1
Injection temperature: 280 °C
Oven temperature programme: initial 40 °C for 2 min; rate 20 °C/min; final 280 °C for 15 min
Column head pressure: 2.0 psi (constant pressure)
Transfer line temperature: 290 °C
MS quad temperature: 150 °C
MS source temperature: 230 °C
Mass range: scan 35 to 200
Injection volume: 1 µL
Retention time of the parent substance: approx. 6.4 min
Retention time of the hydrolysis product: approx. 4.2 min

A library search was performed on the spectrum obtained for the main hydrolysis product peak.
Statistics:
Calculations of sample solution concentrations and reaction kinetics are reported under “Any other information on materials and methods incl. tables”.

Results and discussion

Preliminary studies:
Screening test: there were no significant changes in the infrared scans of the sample solution over time. Therefore, it was not possible to produce an estimate of the rate of hydrolysis of the test substance.
Main ADME results
Type:
other: mean rate constant of hydrolysis in rat plasma (test substance)
Results:
7.83 x 10E-02 min-1, corresponding to a half-life of 8.85 min at 37 ± 0.5 °C

Toxicokinetic / pharmacokinetic studies

Details on absorption:
not applicable
Details on distribution in tissues:
not applicable
Details on excretion:
not applicable

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The identified and expected hydrolysis product of the test item after GC-MS analysis was 2-(1-methylethoxy) ethanol.

Any other information on results incl. tables

CONCENTRATION OF THE SAMPLES AND DATA USED FOR THE CALCULATION OF RATE CONSTANT AND HALF-LIFE

A) DEFINITIVE TEST 1

During test 1, the time points chosen were to widely spaced as the rate of hydrolysis was more rapid than expected, with no test item being detected in any except the initials sample. Therefore, data from test 1 was not sufficient to calculate a result and 3 further tests were performed with the time points taken much more frequently, with dilution and filtration being performed as quickly as possible.

B) DEFINITIVE TEST 2

The following rate constant and half-life for hydrolysis were calculated based on the determined concentrations of the test substance after different incubation time points (see Table 2):

Slope = -9.87 x 10E-02 of linear regression graph

R² = 0.999 of linear regression graph

k = 9.87 x 10E-02 min-1

t1/2 = 7.02 min

 

Table 2. Concentration of the test substance in rat plasma after different incubation time points

Timepoint (min)

Concentration (mg/mL)

Ln C

0

862

6.76

15

176

5.17

30

40.4

3.70

45

10.1

2.31

Ln C = natural logarithm of the test substance concentration

 

C) DEFINITIVE TEST 3

The following rate constant and half-life for hydrolysis were calculated based on the determined concentrations of the test substance after different incubation time points (see Table 3):

Slope = -6.80 x 10E-02 of linear regression graph

R² = 0.9902 of linear regression graph

k = 6.8 x 10E-02 min-1

t1/2 = 10.2 min

 

Table 3. Concentration of the test substance in rat plasma after different incubation time points

Timepoint (min)

Concentration (mg/mL)

Ln C

0

1.12E+03

7.02

8

586

6.37

16

324

5.78

24

191

5.25

32

98.0

4.58

40

61.5

4.12

48

46.8

3.84

Ln C = natural logarithm of the test substance concentration

 

D) DEFINITIVE TEST 4

The following rate constant and half-life for hydrolysis were calculated based on the determined concentrations of the test substance after different incubation time points (see Table 4):

Slope = -6.81 x 10E-02 of linear regression graph

R² = 0.989 of linear regression graph

k = 6.81 x 10E-02 min-1

t1/2 = 10.2 min

 

Table 4. Concentration of the test substance in rat plasma after different incubation time points

Timepoint (min)

Concentration (mg/mL)

Ln C

0

1.04E+03

6.95

8

461

6.13

16

283

5.65

24

188

5.24

32

112

4.72

40

70.3

4.25

48

30.6

3.42

Ln C = natural logarithm of the test substance concentration

 

E) RESULTS FROM TESTS 2, 3 and 4

Mean rate constant (k) of hydrolysis = 7.83 x 10E-02 min-1 (standard deviation: 1.77 x 10E-02)

Mean half-life (t1/2) = 8.85 min

During the tests, the samples were prepared at a nominal concentration of 1400 mg/L. However, analysis of the initial samples gave concentrations from 862 to 1.12E+03 mg/L. This has been attributed to loss of test item through hydrolysis during test preparation. As all times for subsequent time points were relative to the time, the initial samples were treated and the correlation of all plots were good, this was considered to have negligible impact on the results.

 

VALIDATION OF THE ANALYSIS METHOD (GC)

The linearity of the detector response with respect to concentration of the test item was assessed over the concentration range of 5 to 250 mg/L. This was satisfactory with a correlation coefficient (r) of 1.000 being obtained.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results