Reaction mass of ethane-1,2-diol and 2-hydroxyethyl formate and ethylene diformate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

Biotransformation in intestinal-fluid simulant

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

other: Note for Guidance for Food Contact Materials, Annex 1 to Chapter III

Principles of method if other than guideline:

Biotransfromation in intestinal-fluid simulant

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of ethane-1,2-diol and 2-hydroxyethyl formate and ethylene diformate

EC Number:

944-390-9

Molecular formula:

not applicable, multiconstituent substance

IUPAC Name:

Reaction mass of ethane-1,2-diol and 2-hydroxyethyl formate and ethylene diformate

Test material form:

liquid

Administration / exposure

Details on study design:

The test material was dissolved in demineralized water. An aliquot of the solution was transferred to the intestinal-fluid simulant, which was maintained one hour at 37°C with continual agitation. The concentration of the test material in the intestinal fluid-simulant was around 1200 mg/l. After a specified time period the concentrations of ethylene glycol (EG), ethylene glycol monoformate (EGM) and ethylene glycol diformate (EGD) were determined in the hydrolysates via gas chromatography (GC) with a mass selective detector (MS) and quantified using an external standard method composed of five calibration levels. The hydrolysis degree of ethylene glycol monoformate (EGM) and ethylene glycol diformate (EGD) in the intestinal-fluid simulant were calculated. The measurement of hydrolysis in the intestinal-fluid simulant was performed in triplicate.

Preparation of the intestinal fluid simulant
6.800 g of potassium dihydrogen orthophosphate (KH2PO4) were weighed in a 1 litre beaker and dissolved in 250 ml of water. 190 ml of 0.2 M sodium hydroxide (NaOH) were added. The mixture was then shaken. 300 ml of water were added. The mixture was vigorously shaken.
9.994 g of pancreatin extract were weighed into a 200 ml beaker. Ca. 20 ml water were added. The mixture was stirred to make a homogenous paste. Gradually the paste was diluted with small portions of water, and well stirred after each dilution, to give approximately 150 ml of a suspension. The suspension was transferred to the 1 litre beaker. The 200 ml beaker and the funnel were rinsed with water. 0.4983 g of sodium taurocholate were added. The 1 liter beaker was then vigorously shaken. The pH of the solution was 7.13 and was adjusted to 7.55 with a 0.2 M sodium hydroxide solution. The suspension was transferred to a 1 litre volumetric flask. The flask was then filled up to the mark with water.

Details on dosing and sampling:

Hydrolysis test
50.0 ml of the intestinal-fluid simulant were introduced into a 50 ml flask. The test samples were warmed up in a thermostatic cabinet at 37°C during one hour with continual agitation. This warm-up phase was followed by the hydrolysis study at 37°C for 4 hours maximum. 0.5 ml of the test item stock solution were injected into the 50 ml flask with a pipette. The test samples were placed for 1, 2 or 4 hours at 37°C under continual agitation or analysed immediately.
After homogeneization the solution was filtered through a 30 mm syringe filter (0.2 μm nylon). 150 μl of the filtered solution were pipetted into a 1.5 ml vial and mixed with 1.35 ml acetone before injection into the GC-MS. The measurement of hydrolysis in the intestinal fluid simulant was performed in triplicate.

Statistics:

The hydrolysis rate constant and the half-life time of ethylene glycol diformate were determined by linear regression of ln(CEGD/CEGD,t=0) versus time [h]. Analysis of the initial time point indicated that the test item had started to hydrolyse prior to analysis.

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

ethane-1,2-diol and formic acid

Any other information on results incl. tables

Hydrolysability of ethylene glycol monoformate and ethylene gylcol diformate in intestinal-fluid simulant

Time [h]	EGM hydrolysis degree HEGM[%]	EGD hydrolysis degree HEGD[%]
0 (measured)	6.4	9.1
1	10.2	81.8
2	33.6	95.1
4	61.4	99.6

The concentration of each substance at the beginning of hte hydrolysis study in the intestinal-fluid simulant and at specified time periods.

Time [h]	EG concentration [mg.l^-1]	EGM concentration [mg.l^-1]	EGD concentration [mg.l^-1]
0 (calculated)	171.4	580.0	421.8
0 (measured)	147.7	543.1	383.6
1	382.6	521.0	76.6
2	504.7	385.0	20.6
4	644.0	223.7	1.6*

* Value outside the calibration range

The hydrolysis rate constant and the half-life time of ethylene glycol diformate were determined by linear regression of ln(C(EGD)/C(EGD,t=0)) versus time [h]. Analysis of the initial time point indicated that the test item had started to hydrolyse prior to analysis.

At the beginning, the hydrolysis of ethylene glycol monoformate was observed, but also its formation as degradation product of ethylene glycol diformate. This could explain that the observed regression of ln(C(EGM)/C(EGM,t=0)) versus time [h] was not linear. After 1 hour,

81.8% of ethylene glycol diformate were hydrolysed. If it is assumed that at this time the EGM concentration was at the maximum, the hydrolysis rate constant and the half-life time of ethylene glycol monoformate can be determined by linear regression of ln(C(EGM)/C(EGM,t=1)) versus time [h].

The rate constants and half-life times of the hydrolysis of ethylene glycol diformate and ethylene glycol monoformate at 37°C in the intestinal-fluid simulant were calculated.

	Rate constant k [h^-1]	Half-life time t1/2 [h^-1]
Ethylene glycol diformate	1.360	0.35
Ethylene glycol monoformate	0.277	2.40

Where

ln0.5 = -1.3596 x t1/2(EGD) - 0.2236

ln0.5 = -0.2774 x (1 + t1/2(EGM)) + 0.2508

Applicant's summary and conclusion

Conclusions:

Ethylene glycol diformate has a rate constant of k= 1.360 h-1 and a half-life of t1/2 = 0.35 h
Ethylene glycol monoformate has a rate constant of k= 0.277 h-1 and a half-life of t1/2 = 2.40 h

Executive summary:

The biotransformation of the reaction mass was investigated in an intestinal-fluid simulant. The reaction mass was incubated at 37°C at pH 7.5 for 4 hours. After 0, 1, 2 and 4 hours of incubation the concentrations of ethylene glycol (EG), ethylene glycol monoformate (EGM) and ethylene glycol diformate (EGD) were determined in the hydrolysates via gas chromatography (GC) with a mass selective detector (MS) and quantified using an external standard method composed of five calibration levels. The hydrolysis degree of ethylene glycol monoformate (EGM) and ethylene glycol diformate (EGD) in the intestinal-fluid simulant were calculated. The measurement of hydrolysis in the intestinal-fluid simulant was performed in triplicate.

The rate constant k and the half-life t1/2 were k= 1.360/h and t1/2 = 0.35 h for ethylene glycol diformate and k= 0.277/h and t1/2 = 2.40 h for ethylene glycol monoformate.

The results of the study demonstrate rapid transformation of ethylene glycol diformate into ethylene glycol monoformate and formic acid and further transformation of ethylene glycol monoformate into ethan-1,2-diol and formic acid.