Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27 Jul - 29 Aug 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. No hydrolysis test in saliva and gastric juice simulants.

Data source

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

Materials and methods

Objective of study:
other: hydrolysis in intestinal fluid simulant
Test guideline
Qualifier:
according to
Guideline:
other: European Food Standard Authority, Food Contact Materials, Note for Guidance (updated 30/07/2008), Annex 1 to Chapter III
Deviations:
yes
Remarks:
no hydrolysis test in saliva and gastric juice simulants
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): Ethylene Glycol Diacetate
- Physical state: clear colourless liquid
- Analytical purity: 99.72%
- Lot/batch No.: 4180
- Expiration date of the lot/batch: 18 April 2014
- Storage condition of test material: at room temperature in the dark
Radiolabelling:
no

Test animals

Species:
other: not specified; presumably pig in accordance with the test method used
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
TEST DIGESTIVE SIMULANTS
INTESTINAL FLUID SIMULANT
- Description: Intestinal fluid simulant in accordance with the guideline
- Preparation: Potassium dihydrogen orthophosphate (KH2PO4) (3.41 g) was dissolved in approximately 125 mL of reverse osmosis (RO) water, in a 500 mL volumetric flask. Approximately 95 mL of 0.2 M sodium hydroxide (NaOH) was added followed by a further 200 mL of RO water. Pancreatic extract (5.00 g) was weighed into a 150 mL beaker and approximately 75 mL of RO water added gradually with stirring. The solution was then transferred to the volumetric flask containing the basified KH2PO4. Sodium taurocholate (0.25 g) was added and the mixture shaken gently. The pH of the solution was adjusted to pH 7.5 ± 0.1 using 0.2 M NaOH. The solution was diluted to volume using RO water and shaken thoroughly to mix.

Administration / exposure

Route of administration:
other: mixing
Vehicle:
unchanged (no vehicle)
Details on exposure:
- Preparation of the Test Solutions
Sample solutions were prepared in capped glass vessels at a nominal concentration of 1.5 g/L (equivalent to approximately 0.01 mol/L) in the intestinal fluid simulant.
The test solutions were split into individual vessels for each data point.
The solutions were shielded from light whilst maintained at the test temperature.

- Testing
The sample solutions were maintained at 37.0 ± 0.5 °C for a period of 1, 2 and 4 h.
Duration and frequency of treatment / exposure:
1, 2 and 4 h
Doses / concentrations
Remarks:
Doses / Concentrations:
1.5 g/L (equivalent to approximately 0.01 mol/L)
No. of animals per sex per dose:
triplicate determinations
Control animals:
other: for GC: blank samples from intestinal fluid simulant and samples of reference materials (parent substance and hydrolysis products), all diluted 1:10 in acetone
Details on study design:
- Principle of the Test
The test item was dissolved in the requisite test medium and the solution incubated in the dark and at 37.0 ± 0.5 °C in a water bath. The concentration of the test item was determined as a function of time, using a suitable analytical method. For decreasing concentrations, the logarithms of the concentrations were plotted against time (log10 (Ct)). For plots resulting in a straight line, the reaction is considered to be of (pseudo-) first order. The rate constant and the half-time are then calculated using the slope.
Details on dosing and sampling:
- Analysis of the Sample Solutions
Triplicate sample solutions 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).

- Controls
Standards: Duplicate standard solutions of a mixture of test item, acetic acid and ethanediol were prepared in acetone at nominal concentration of 150 mg/L each.

Samples: An aliquot of each sample solution was diluted by a factor of 10 using acetone.

Matrix blanks: Intestinal fluid simulant diluted by a factor of 10 using acetone.

- Analysis
The standard and sample solutions were analysed by GC using the following conditions:

GC system: Agilent Technologies 6890, incorporating workstation
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 7min; final 240 °C for 8 min
Injection mode: splitless (purge on at 0.5 min)
Flow rate: 0.75 mL/min
Injection temperature 300 °C
FID detector temperature: 250 °C
Injection volume: 1 µL
Retention time: Acetic acid ca. 9.7 min; Ethanediol ca. 11.4 min; Test item ca. 14.1 min

- Data handling
Sample solution concentration: The response factors of the standard peak areas (unit peak area per mg/L) were calculated using Equation 1:

RF = (Rstd/Cstd) [Equation 1]

Where:
RF = response factor for the standard solution
Rstd = peak area for the standard solution
Cstd = concentration for the standard solution (mg/L)

The sample solution concentration (g/L) was calculated using Equation 2

C = (Rspl/RFstd) x (D/1000) [Equation 2]

Where:
C = sample solution concentration (g/L)
Rspl = mean peak area for the sample solution
RFstd = mean response factor for the standard solutions (unit peak area per mg/L)
D = dilution factor (10)

- Degree of Hydrolysis
The decrease in concentration of test item or the degree of hydrolysis was calculated using Equation 3.

Degree of hydrolysis in percent = ((C0 – Ct)/C0) x 100 [Equation 3]

Where:
C0 = concentration at time 0
Ct = concentration at time t

- Half-life
For confirmation of first order kinetics, the data obtained in the tests were plotted as log10 (Ct) versus t.
From this plot the reaction rate constant (Kobs) was obtained from the slope, where the slope of the resultant line was calculated using linear regression analysis as per Equation 4.

Kobs = -2.303 x slope [Equation 4]

The half-life time of the reaction was calculated using Equation 5.

t1/2 = 0.693/Kobs [Equation 5]
Statistics:
Mean values of triplicate determinations were calculated.

Results and discussion

Main ADME results
Type:
other: degree of hydrolysis
Results:
4.37, 6.42 and 10.5% after 1, 2 and 4 h, respectively

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 25.9 h at 37 °C
Test no.:
#1
Toxicokinetic parameters:
other: rate constant = 7.42E-06 per sec

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Acetic acid concentration was determined in the initial (0 h) and in the 1, 2, and 4 h samples.
The corresponding mean concentrations were 74.9, 220, 261 and 423 mg/L at 0, 1, 2 and 4 h, respectively.
Ethanediol concentrations were not determined.
A peak in the chromatography at approximately 12.7 minutes, which was not present in the standard solutions, was observed to increase in magnitude over the period of the test. This was not identified or quantified but was considered most likely to be ethylene glycol monoacetate, a degradation product of the test item.

Any other information on results incl. tables

Table 1. Degree of hydrolysis

Sample

Sample pH (mean of 3 samples)

Mean concentration (mg/L), n=3

Log10 Concentration (mg/L)

Degree of hydrolysis (%)

Initial

7.45

1503

3.177

-

1 h

7.36

1438

3.158

4.37

2 h

7.35

1407

3.148

6.42

4 h

7.27

1345

3.129

10.5

 

Table 2. Results of linear regression

Slope

-1.16E-02

Kobs

2.67E-02 per h (7.42E-06 per sec)

t1/2

25.9 h (1.08 days)

 

Table 3. Acetic acid concentration

Sample

Mean concentration (mg/L), n=3

Initial

74.9

1 h

220

2 h

261

4 h

423

 

 

Validation

The linearity of the detector response with respect to concentration was assessed over the concentration range of 15 to 300 mg/L for both test item an acetic acid. This was satisfactory with correlation coefficients (r) of 0.999 being obtained for each analyte.

 

Discussion

It was observed that the concentration of acetic acid in the sample solutions increased with time and was most likely due to hydrolysis of the test item.

 

A peak in the chromatography at approximately 12.7 minutes, which was not present in the standard solutions, was observed to increase in magnitude over the period of the test. This was not identified or quantified but was considered most likely to be ethylene glycol monoacetate, a degradation product of the test item.

 

Conclusion

The rate constant and half-life at 37 °C of the test item in simulated intestinal fluid at pH 7.5 were determined and are shown below.

 

Table 4. Rate constant and half-life time

Rate constant (per sec)

Estimated half-life at 37 °C

7.42E-06

25.9 h

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results