Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions (only one intestinal fluid simulant tested)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EFSA Note for Guidance for Food Contact Materials (updated 30 Jul 2008), Annex 1 to Chapter III
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 111 (Hydrolysis), 13 Apr 2004
Deviations:
yes
Remarks:
(tested in intestinal fluid simulant)
GLP compliance:
yes (incl. QA statement)
Remarks:
(Department of Health, London, UK)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene diformate
EC Number:
211-077-7
EC Name:
Ethylene diformate
Cas Number:
629-15-2
Molecular formula:
C4H6O4
IUPAC Name:
2-(formyloxy)ethyl formate
Details on test material:
- Name of test material (as cited in study report): Ethylene Glycol Diformate
- Physical state/appearance: extremely pale yellow liquid
- Analytical purity: > 80% w/w
- Batch No.: 13082
- Expiry Date: 28 Jun 2013
- Storage conditions: room temperature in the dark
Radiolabelling:
no

Test animals

Species:
other: simulated intestinal fluid
Strain:
other: not applicable (in vitro)
Sex:
not specified
Details on test animals or test system and environmental conditions:
INTESTINAL FLUID SIMULANT
- Preparation: 6.8 g KH2PO4 was dissolved in approx. 250 mL of reverse osmosis (RO) water. Approx. 190 mL of 0.2 M NaOH was added followed by a further 400 mL of RO water. Pancreatin extract (10 g) was dissolved in approx. 150 mL of RO water and transferred to the basified KH2PO4. Sodium taurocholate (0.5 g). The pH was adjusted to 7.5 ± 0.1 with 0.2 M NaOH. The solution was diluted to volume using RO water.

Administration / exposure

Route of administration:
other: mixing
Vehicle:
other: simulated intestinal fluid
Details on exposure:
- Preparation of the test solutions:
The test item was diluted to 500 mL using simulated intestinal fluid to a concentration of 1.51E03 mg/L, equivalent to 0.01 mol/L.
The solutions were shielded from light whilst maintained at the test temperature.
Triplicate sample solutions (A, B and C) were taken from the water bath after the specified incubation period and the pH of each solution recorded. The concentration of the sample solution was determined by gas chromatography (GC).
- Preparation of internal standard solution:
Duplicate standard solutions of a mixture of test item, formic acid and ethanediol were prepared in acetone at nominal concentrations of 150 mg/L each.
- Preparation of sample solution:
An aliquot of each sample solution was diluted by a factor of 10 using acetone.
- Matrix Blank:
Intestinal fluid simulant diluted by a factor of 10 using acetone.

Standards, samples and matrix blank were centrifuged (13500 rpm, 15 min) to remove any undissolved material which may have originated from the simulated intestinal fluid, prior to vialing for analysis.
Duration and frequency of treatment / exposure:
1, 2 and 4 h at 37 °C
Doses / concentrations
Remarks:
Doses / Concentrations:
1.51 mg/mL
No. of animals per sex per dose / concentration:
triplicate determination
Control animals:
other: for GC: blank samples from test digestive simulants and samples of reference materials (parent substance and hydrolysis products)
Details on dosing and sampling:
Details on analysis by GC:
- GC System: Agilent Technologies 6890
- Column: DB-624 (60 m x 0.25 mm id x 1.4 µm film)
- Oven temperature program: initial 40 °C for 2 min; rate 10°C/min; final 80 °C for 0 min; rate 20 °C/min; final 240 °C for 8 min
- Injection mode: splitless
- Flow rate: 0.75 mL/min
- Injection temperature: 300 °C
- FID detector temperature: 250 °C
- Injection volume: 1 µL
- Retention time: ethanediol approx. 12.0 min; test item approx. 13.5 min

DATA HANDLING - EQUATIONS
Sample Solution Concentration:
- Response factor for the standard solution = peak area or height for the standard solution/concentration for the standard solution (mg/L)
- Sample solution concentration (g/L) = (mean peak area or height for the sample solution/mean response factor for the standard solutions)/(Dilution factor: 10/1E03)
Degree of Hydrolysis:
- Degree of hydrolysis in percent = ((concentration at time 0 - concentration at time t)/concentration at time 0) x 100
Half-Life:
- Half-life time : 0.693/Kobs (Kobs (reaction rate constant): -2.303 x slope of linear regression)
Statistics:
Mean values of triplicates were calculated.

Results and discussion

Main ADME resultsopen allclose all
Type:
other: ester hydrolysis in intestinal fluid simulant (test substance)
Results:
79.7%, 91.4% and 100% after 1, 2 and 4 h, respectively
Type:
other: ester hydrolysis in intestinal fluid simulant (test substance)
Results:
31% at initial measurement

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:
Ethanediol: The increase in concentration of ethanediol with time was demonstrated via GC.

Any other information on results incl. tables

Table 1: pH value and test item concentrations at the given time points

Sample

Mean peak area

pH value

Conc. test item (mg/L)

Mean conc. (mg/L)

Log10 conc. (mg/L)

Degree of Hydrolysis* (%)

Test item standard 151 mg/L

6.752E04

-

-

-

-

-

Test item standard 159 mg/L

8.112E04

-

-

-

-

-

Initial Sample A

4.892E04

7.51

1020

1030

3.01

31.6

Initial Sample B

4.919E04

1030

Initial Sample C

5.013E04

1050

1-Hour Sample A

1.569E04

6.95

327

306

2.49

79.7

1-Hour Sample B

1.245E04

6.96

260

1-Hour Sample C

1.591E04

6.94

332

2-Hour Sample A

5.457E03

6.88

114

130

2.11

91.4

2-Hour Sample B

8.938E03

6.88

186

2-Hour Sample C

4.331E03

6.88

90.4

4-Hour Sample A

No peak

6.85

Non detected

Non detected

-

100

4-Hour Sample B

No peak

6.83

Non detected

4-Hour Sample C

 No peak

6.82

Non detected

* Degree of Hydrolysis has been calculated using the test item stock theoretical concentration (1.51E03 mg/L).

 

The following parameters were determined by linear regression analysis of Log10(test concentration) (mg/L) vs. time (hours)

Slope: -0.449

kobs: 1.03 1/hour = 2.87E-04 1/second = 40.2 minutes

t(1/2): 0.67 hours

 

Table 2:

The ethanediol concentrations at the given time points

Sample

Mean peak area

Conc. of test item (mg/L)

Mean conc. (mg/L)

Ethanediol standard 182 mg/L

5.664E05

-

-

Ethanediol standard 151 mg/L

4.896E05

-

-

Initial Sample A

No peak

Non detected

Non detected

Initial Sample B

No peak

Non detected

Initial Sample C

No peak

Non detected

1-Hour Sample A

4.633E04

146

158

1-Hour Sample B

4.957E04

156

1-Hour Sample C

5.492E04

173

2-Hour Sample A

1.000E05

314

338

2-Hour Sample B

1.049E05

330

2-Hour Sample C

1.279E05

371

4-Hour Sample A

1.283E05

403

428

4-Hour Sample B

1.344E05

423

4-Hour Sample C

1.460E05

459

The rate constant and half-life at 37 °C of the test item in sumulated intestinal fluid at pH 7.5 were determined:

Rate constant: 2.87E-04 1/s

Estimated half-life at 37 °C: 0.67 hours

Analysis of the initial time point indicated that the test item had started to hydrolyse prior to analysis. Therefore, the degree of hydrolysis has been calculated based on the theoretical stock concentration. It was observed that the concentration of ethanediol in the sample solutions incerased with time and was most likely due to hydrolysis of the test item. Peaks in the chromatography at approx. 9.3 and 13.0 minutes, which were not present in the standard solutions, were observed to increase in magnitude over the period of the test. These were not identified or quantified but were considered most likely to be test item degradation. Mixed standards were prepared containing test item, formic acid and ethanediol. However, formic acid was found not to give a peak under the GC conditions used in this study. Since there was no measurable peak for the test item in the 4-hour sample time point, the rate constant and half-life have been caluclated using results from the initial, 1-hour and 2-hour samples.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: rapid hydrolysis
Full hydrolysis within 4 hours of the test item in intestine fluid simulant at a pH value of 7.5 at 37 °C was demonstrated in this hydrolysis study. The half-life in simulated intestinal fluid was calculated to be 0.67 hours.