Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 - 11 Mar 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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:

equivalent or similar to guideline

Guideline:

other: EFSA Note for Guidance for Food Contact Materials Annex 1 to Chapter III MEASUREMENT OF HYDROLYSIS OF PLASTICS MONOMERS AND ADDITIVES IN DIGESTIVE FLUID SIMULANTS (30/07/2008)

Deviations:

yes

Remarks:

no hydrolysis test in saliva and gastric juice simulants

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, monoester with oxybis(propanediol)

EC Number:

256-367-4

EC Name:

Oleic acid, monoester with oxybis(propanediol)

Cas Number:

49553-76-6

Molecular formula:

C24H46O6

IUPAC Name:

3-(2,3-dihydroxypropoxy)-2-hydroxypropyl octadec-9-enoate

Radiolabelling:

no

Test animals

Species:

other: pancreatin from porcine pancreas

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

INTESTINAL FLUID SIMULANT
- Description: the intestinal fluid simulant contains pancreatin from porcine pancreas as hydrolytic catalyst.
- Preparation: reported to have been done according to the guideline.
- Source of Pancreatin: SIGMA P7545 Lot SLBD06040V; 8 x USP; CAS: 8049-47-6

Administration / exposure

Route of administration:

other: mixing

Vehicle:

other: tetrahydrofuran (THF) (presolution)

Details on exposure:

PREPARATION OF INTERNAL STANDARD SOLUTION:
An internal standard solution with a concentration of approx. 0.6 mg/g n-heptadecane (C17 n-alkane; Aldrich Chemistry) in heptane:pyridine (vol. 2:1) was prepared.

PREPARATION OF BLANK SAMPLE:
A 100 mL sample of intestinal fluid simulant was extracted (see “Details on dosing and sampling”).

PREPARATION OF TMS REAGENT FOR SILYLATION DERIVATISATION:
N, O bis-trimethylsilyl trifluoro-acetamide (BSTFA) was mixed 100:1 (v/v) with trimethyl chlorosilane (TMCS) for preparation of the TMS reagent.

PREPARATION OF CALIBRATION SAMPLE:
50 mg of the calibration sample (trade name) was weighted into a glass container with screw cap, 12 mL of internal standard solution was added and the container was tightly closed and reweighed. After mixing, 500 µL of the sample solution was transferred to a crimp vial and 100 µL TMS reagent was added. Then, the crimp vial was closed using a TEFLON® coated seal and mixed well. The vial was placed in a thermostatic heating block for 15 min at 60 °C. Approx. 1 µL of the sample solution was injected into the GC system

HYDROLYSIS WITH INTESTINAL-FLUID SIMULANT:
For the hydrolysis investigation, 50 mg of test item was dissolved in 2 mL tetrahydrofuran (THF). THF was gently removed in a stream of compressed air and a thin film of the product was formed on the bottom of the flask. 50 mL of simulant without enzyme was added and the test substance was dispersed in the aqueous phase while stirring for 2 min by magnetic stirring. Samples were taken after 0, 1, 2 and 4 h.

Duration and frequency of treatment / exposure:

0, 1, 2 and 4 h

Doses / concentrations

Dose / conc.:

50 other: mg

No. of animals per sex per dose / concentration:

not applicable, the test was performed in triplicates

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:

EXTRACTION OF HYDROLYSATES
1. 1 mL of formic acid was added to the 250 mL Bluecap flask containing the hydrolysate while stirring.
2. 50 mL of methyl tert-butyl ether (MTBE) was added and the stirring continued for 2 min.
3. 25 mL of P-ether (40-60) was added and the stirring continued for 15 sec.
4. While stirring 10-20 mL of 96% ethanol was added and the stirring was stopped.
5. The phase separation was improved by very gently adding up to 10 mL ethanol to the organic upper phase.
6. The clear upper phase was transferred to a 250 mL round bottomed flask.
7. Steps 2-6 were repeated twice.
8. The solvent was removed by means of a rotatory evaporator (approx. 70 °C, 15 mmHg)
9. 12 mL of internal standard (Heptadecane) solution was weighed into the round-bottomed flask containing the dried hydrolysate.
10. A 500 mL aliquot of the solution was transferred to a 2 mL crimp vial and 100 µL TMS reafgen was added.
11. The crimp vial was sealed and heated to 60 °C for 15 min.
12. A 1 µL aliquot was injected into the GC system by means of an auto sampler.

DETERMINATION OF HYDROLYSIS PRODUCTS (GC ANALYSIS)
- Principle:
The hydrolysis products of the test substance were extracted by means of tetrahydrofuran (THF), methyl tert-butyl ether (MTBE), petroleum ether (bp. 40-60 °C) and ethanol (96%). Formic acid was added prior to extraction to convert fatty acid salt (soaps) into free fatty acids. Free hydroxyl groups and free acids in the extract were protected by means of trimethyl silyl groups (silylation) and non-hydrolysed diglycerol monooleate was quantified by means of GC.
- Calibration standard:
A sample of pure test material was analysed.
- Internal standard:
The calibration was carried out by an internal standard calibration procedure. The internal standard was n-Heptadecane (C17 n-alkane).
- Apparatus:
GC instrument: Perkin Elmer Clarus 600 equipped with an autosampler, FID detector and a programmable split/splitless injector operated in the cold split mode.
GC Integration system: Perkin Elmer Turbochrom Workstation ver. 6.3.2.0646
- Blank sample preparation:
To identify any blank peaks in the GC chromatograms100 ml samples of intestinal fluid simulant was extracted and analysed by means of GC.
- Optimisation of instrumentation:
The GC system was optimised for analysis of silylated glycerides. This was carried out by optimising the instrumental conditions in order to comply with the repeatability values given for analysis of glycerides in mono- and diglyceride concentrates given in American Oil Chemists’ Society, Champaign Illinois; Official Methods and Recommended Practices of the AOCS 5 th. ed.; Official Method Cd 11b-91: “Determination of Mono- and Diglycerides by Capillary Gas Chromatography”.
- Calculation of components.
Calculation of the content of hydrolysis components in the sample was carried out using the formula:
w/w% component = [Response factor (component) x Area (component) x mg Internal standard x 100%] / [Area (Internal standard) x mg Sample prior to hydrolysis]

- Determination of response factors:
The Response factor for the main component of the hydrolysis products (= parent substance), inclusing all diglcerol monooleate isomers, was determined experimentally relative to n-heptadecane. The Response factor (Rf)for a retention time range of main components (X) in the chromatogram was calculated using the following formula:
Rf (X) = [Area (Internal standard) x mg Calibration Sample] / [Area (x) x mg Internal Standard], where mg Internal Standard is calculated from: mg Internal standards (IS) = IS solution (g) x concentration of IS solution (F) [mg/g C17 alkane]

CONFIRMATION ASSAYS
The identification of all components was carried out by comparing retention times of peaks in chromatograms.

RECOVERY
The overall recovery of the method was determined by a simple recovery experiment. The test substance was analysed in triplicate according to the analytical method using a sample of intestinal fluid without enzyme added and with 0 h of hydrolysis. A mean recovery of 95.8% was found (see table 2 under “Any other information on results incl. tables”).

Statistics:

Mean values of triplicates were calculated.

Results and discussion

Main ADME results

Type:

other: ester hydrolysis in intestinal fluid simulant

Results:

84, 90, and 94.3 % 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

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

Table.1 Hydrolysis of the test item with intestinal-fluid simulant

Hydrolysis study
Concentration of internal standard (I.S.) solution (F): 0.6422 mg/g C17 alkane
Experiment No.	1535	1536	1537	1539	1540	1541	1545	1546	1547	1548	1549	1550
Time of hydrolysis	0 hours			1 hours			2 hours			4 hours
Data:
mg sample	51.80	49.31	47.10	51.34	48.26	48.16	53.46	49.25	50.50	51.77	52.73	49.98
I.S. solution (g)	9.5952	9.7220	9.5071	9.5686	9.4879	9.4755	9.3912	9.5178	9.5252	9.5616	9.5043	9.4770
mg I.S (calculated)	6.162	6.243	6.106	6.145	6.093	6.083	6.031	6.112	6.117	6.1414	6.104	6.086
Area (I.S.)	336862	352376	335097	338291	340040	340192	337381	342221	340950	344348	358211	338235
Area (X)	1892921	1891083	1863661	368325	369106	339607	247633	229627	225207	132125	149457	132971
Rf	1.226	1.226	1.226	1.218	1.218	1.214	1.214	1.214	1.214	1.216	1.216	1.216
Results:
% test item	81.95	83.31	88.39	15.87	16.69	15.36	10.05	10.11	9.71	5.53	5.87	5.82
Mean	84.6			16.0			10.0			5.7

 

Table 2. Recovery experiment (0 hours without enzyme added)

Recovery study
Concentration of internal standard solution (F): 0.6422 mg/g C17 alkane
Experiment No.	1542	1543	1544
Time of hydrolysis	0 hours
Data:
mg sample	47.27	50.36	50.48
Internal standard solution (g)	9.4822	9.4909	9.4631
mg internal standard solution (calculated)	6.090	6.095	6.077
Area (internal standard)	330728	340028	334857
Area (X)	2036744	2227788	2201882
Rf	1.209	1.209	1.209
Results:
% PGE O80	95.91	95.87	95.71
Mean	95.8

Applicant's summary and conclusion

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results