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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (no data on GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Objective of study:
other: hydrolysis in digestive fluid simulants
Test guideline
Qualifier:
according to
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
Deviations:
no
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Description: fully acetylated monoglyceride based on fully hydrogenated castor oil
- Analytical purity: > 83% (test material); 86.59% (calibration, in-house standard for GC)
- Purity test date: 26 Feb 2002 (GC standard)
- Lot/batch No.: 10102
- Storage condition of test material: in a refrigerator/desiccator
Radiolabelling:
no

Test animals

Species:
pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
TEST DIGESTIVE SIMULANTS
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 10K1642; 8 x USP SIGMA specifications: “Contains many enzymes, including amylase, trypsin, lipase, ribonuclease and protease.” CAS: 8049-47-6

SALIVA SIMULANT
- Description: carbonate buffer with a pH value of 9
- Preparation: reported to have been done according to the guideline.

GASTRIC JUICE SIMULANT
- Description: 0.07 M hydrochloric acid
- Preparation: reported to have been done according to the guideline.

Administration / exposure

Route of administration:
other: mixing
Vehicle:
other: tetrahydrofuran (THF)
Details on exposure:
- Preparation of internal standard solution
1. 5.8 g n-heptadecane was accurately weight to a 25 mL measuring flask.
2. The flask was filled to the mark with tetrahydrofuran (THF) and the amount of THF was determined by weighing.

- Preparation of sample solution
1. 18.75 g of test material or positive control was weight to a 25 mL measuring flask.
2. The measuring flask was filled to the mark with internal standard solution and the amount of internal standard solution was determined by weighing.

- In vitro hydrolysis of sample solution
1. 100 µL of the sample solution was transferred to a 100 mL erlenmeyer flask.
2. The digestive fluid hydrolysis was carried out according to the guideline (see Duration and frequency of treatment / exposure).
Duration and frequency of treatment / exposure:
Intestinal fluid simulant: 1, 2 and 4 h
Saliva simulant: 0.5 h
Gastric juice simulant: 1, 2 and 4 h
Doses / concentrations
Remarks:
Doses / Concentrations:
750 µg/mL
No. of animals per sex per dose:
triplicate determinations
Control animals:
other: for GC: blank samples from test digestive simulants and samples of reference materials (parent substance and hydrolysis products)
Positive control:
Sunflower oil (oleic acid content ca. 80%; triglyceride content ca. 99% (GC))
Details on dosing and sampling:
DETERMINATION OF HYDROLYSIS PRODUCTS
- Principle:
The hydrolysis products of the test substance were extracted by means of methyl tert-butyl ether (MTBE). Free hydroxyl groups and free acids in the extracted derivatives of 12-hydroxy stearic acid were protected by means of trimethyl silyl groups (silylation) and quantified by means of GC.
- Calibration standard:
The quantification of 12-hydroxystearic acid derivatives was carried out by means of an in-house standard of the test substance. The composition of the in-house standard had been established by means of calibration material obtained by preparative HPLC of the test substance.
- Internal standard:
The calibration was carried out by means of an internal standard calibration procedure. The internal standard was n-Heptadecane (C17 n-alkane) purum; > 98% (GC); Fluka Chemie AG.
- Apparatus:
GC instrument: Perkin Elmer XL Autosystem 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. 6.1.1.0.0.
- Blank sample preparation:
To identify any blank peaks in the GC chromatograms100 ml samples of all tree digestive fluid simulants were 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) relative to n-heptadecane was determined experimentally by means of an in house standard. The relative Response factors for all other components were calculated theoretically based on the effective carbon number concept (ECN concept) described by James T. Scanlon and Donald E. Willis (Chromatogr. Sci. 23(1985); p.333-340). In order to compensate for discrimination, the theoretically calculated response factors of partial glycerides were corrected with the experimental discrimination factor for the parent substance.

CONFIRMATION ASSAYS
- Identification of chromatograms of hydrolysed test substance:
The identification of all components was carried out by comparing retention times of peaks in chromatograms. The following samples were prepared in laboratory scale for identification purposes:
1. Distilled monoglyceride based on fully hardened castor oil
2. 50% acetylated monoglyceride on fully hardened castor oil
3. Fully acetylated monoglyceride on fully hardened castor oil (= test substance)
4. Commercially available 12-hydroxystearic acid recrystallised from toluene (>98%)
5. Distilled fully acetylated 12-hydroxystearic acid (>98%)

- Identification of chromatograms of hydrolysed sunflower oil (positive control):
The identification of partial glycerides was carried out based on in-house reference material for analysis of mono- and diglycerides.
Statistics:
Mean values of triplicate determinations were calculated.

Results and discussion

Main ADME resultsopen allclose all
Type:
other: ester hydrolysis in intestinal fluid simulant (test substance)
Results:
66.7, 83.0 and 93.6% after 1, 2 and 4 h, respectively
Type:
other: ester hydrolysis in intestinal fluid simulant (positive control)
Results:
90.2, 97.6 and 98.6% after 1, 2 and 4 h, respectively
Type:
other: ester hydrolysis in saliva simulant (test substance)
Results:
no hydrolysis
Type:
other: ester hydrolysis in gastric juice simulant (test substance)
Results:
no hydrolysis

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 following mono-/di-/triesters of glycerol were quantitatively determined (w/w% relative to a non-hydrolysed sample) after 0, 1, 2 and 4 h hydrolysis in intestinal fluid simulant, respectively:
Mono-12-(hydroxy)octadecanoate: 0.0, 0.6, 1.3 and 3.0%
Mono-12-(acetoxy)octadecanoate: 0.0, 0.06, 0.06 and 0.04%
Mono-12-(acetoxy)octadecanoate, monoacetate: 1.2, 2.3, 9.2 and 8.3%
Mono-12-(acetoxy)octadecanoate, diacetate (parent substance): 83.1, 27.7, 14.1 and 5.3%

The following free fatty acids were quantitatively determined (w/w% relative to a non-hydrolysed sample) after 0, 1, 2 and 4 h hydrolysis in intestinal fluid simulant, respectively:
12-(hydroxy)octadecanoic acid: 0.0, 0.17, 0.39 and 1.10%
12-(acetoxy)octadecanoic acid: 0.1, 27.7, 29.2 and 31.5%

Any other information on results incl. tables

GC Interferences

A chromatogram of a blank sample from intestinal fluid simulant was compared with a chromatogram of the hydrolysis products after 2 hours. From the chromatograms it was observed that significant interference was absent for all the analysed components except for free 12-(hydroxy)octadecanoic acid, which eluted together with a component from the intestinal fluid simulant.

As a consequence the reported analytical results for 12-(hydroxy)octadecanoic acid should be considered max. values. Since the interference was of little importance for the overall conclusion of the analysis, there was no attempt to further improve peak separation or to adjust the analytical results in respect to a blind value.

GC chromatograms of blank samples of gastric juice simulant and saliva simulant did not contain peaks which could interfere with the analysis.

 

Tables of Results

Table 1 shows the concentration of components in the test system after enzymatic hydrolysis of the test substance after 0, 1, 2 and 4 hours, respectively. Results are given as a mean of triple determinations.

 

Table 1. Content of 12-hydroxystearic acid derivatives in intestinal fluid simulant as function of time of hydrolysis. All figures are given as weight% relative to a non-hydrolysed sample.

Component

0 h

1 h

2 h

4 h

Ester of glycerol

Mono-12-(acetoxy)octadecanoate, diacetate (parent substance)

83.1

27.7

14.1

5.3

Mono-12-(acetoxy)octadecanoate, monoacetate

1.2

2.3

9.2

8.3

Mono-12-(acetoxy)octadecanoate

0.00

0.06

0.06

0.04

Mono-12-(hydroxy)octadecanoate

0.0

0.6

1.3

3.0

Free fatty acid

12-(acetoxy)octadecanoic acid

0.1

27.7

29.2

31.5

12-(hydroxy)octadecanoic acid

0.00

0.17*

0.39*

1.10*

 

* Results should be taken as max. values since interference with intestinal fluid simulant is predominant for this component.

 

Results in Table 1 show that the test substance was extensively hydrolysed by intestinal fluid simulant. The products available for absorption would thus include glycerol, acetate, 12-(hydroxyl)octadecanoic acid and 12-(acetoxy)octadecanoic acid, of which most of the total 12-(hydroxyl)octadecanoic acid is present as the 12-(acetoxy)octadecanoic acid derivative.

As the main component after 4 hours hydrolysis time was free 12-(acetoxy)octadecanoic acid and the content of glycerol 12-(hydroxy)octadecanoate (monoester) and free 12-(hydroxyl)octadecanoic acid had increased to 3.0% and 1.1%, respectively, it was concluded that the intestinal fluid simulant mainly was active on lipid ester bonds and that it had limited activity towards acetyl-ester bonds.

The hydrolysis of ester bonds between acetic acid and glycerol was not examined. This mechanism was, however, assumed to be identical to the hydrolysis of the food additive E472a, acetic acid esters of mono- and diglycerides.

 

In order to obtain some qualitative information about the relative rate of hydrolysis between the test substance and a standard triglyceride, the enzymatic hydrolysis of a high oleic acid sunflower oil was analysed. Table 2 lists the concentration of hydrolysis products after enzymatic hydrolysis of sunflower oil by the intestinal-fluid simulant after 0, 1, 2 and 4 hours. Results are given as means of triple determinations.

 

Table 2. Content of fatty acid containing components in the intestinal-fluid simulant as a function of time. All results are given as weight% relative to a non-hydrolysed sample.

Component

0 h

1 h

2 h

4 h

Free fatty acids

-

48.7

58.3

66.1

Monoglyceride

-

24.7

29.9

30.0

Diglyceride

-

21.2

13.4

10.1

Triglyceride

ca. 99

9.7

2.4

1.4

 

 

Table 3 lists the composition of lipid extract after hydrolysis of the test substance in saliva simulant after 0 and 0.5 hours, respectively. Results are given as means of triple determinations.

 

Table 3. Content of 12-hydroxystearic acid derivatives and octadecanoic acid in saliva simulant as a function of time of hydrolysis of the test substance. All results are in weight% relative to a non-hydrolysed sample.

Component

0 h

1 h

Ester of glycerol

Mono-12-(acetoxy)octadecanoate, diacetate (parent substance)

83.2

84.3

Mono-12-(acetoxy)octadecanoate, monoacetate

1.2

1.3

Free fatty acid

Octadecanoic acid

0.0

0.0

12-(acetoxy)octadecanoic acid

0.1

0.1

 

From the data in Table 3 it was concluded that saliva simulant did not have any hydrolytic effect on the test substance.

Table 4 lists the composition of lipid extract after hydrolysis of the test substance in gastric juice simulant after 0, 1, 2 and 4 hours, respectively. Results are given as means of triple determinations.

 

Table 4. Content of 12-hydroxystearic acid derivatives and octadecanoic acid in gastric juice simulant as function of time of hydrolysis of the test substance. All results are in weight% relative to a non-hydrolysed sample.

Component

0 h

1 h

2 h

4 h

Ester of glycerol

Mono-12-(acetoxy)octadecanoate, diacetate (parent substance)

83.2

84.8

85.8

85.9

Mono-12-(acetoxy)octadecanoate, monoacetate

1.2

1.6

1.6

1.6

Free fatty acid

Octadecanoic acid

0.0

0.1

0.1

0.1

12-(acetoxy)octadecanoic acid

0.1

0.2

0.1

0.1

 

From the data in Table 4 it was concluded that gastric juice simulant did not have any hydrolytic effect on the test substance at contact times up to 4 hours.

Applicant's summary and conclusion

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