Octanoic acid, monoester with glycerol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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)

Objective of study:

other: hydrolysis in digestive fluid simulants

Qualifier:

according 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

Deviations:

no

GLP compliance:

not specified

Radiolabelling:

no

Species:

pig

Strain:

not specified

Sex:

not specified

Details on test animals or test system 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.

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

Remarks:

Doses / Concentrations:
750 µg/mL

No. of animals per sex per dose / concentration:

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 reference chemical:

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.

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

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

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%

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.

Conclusions:

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

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

11 Jul 2003 - 19 Jan 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

metabolism of the test substance was not evaluated

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD (SD) BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., Quebec, Canada
- Age at study initiation: approx. 7 - 10 weeks
- Weight at study initiation: 260 - 362 g
- Housing: individually in stainless steel wire meshbottomed cages equipped with an automatic watering valve
- Individual metabolism cages: yes, for the high-dose group animals (test group 4)
- Diet: certified commercial laboratory diet (Harlan Teklad #8728CM), ad libitum (except during designated procedures)
- Water: municipal tap water, filtered through a 5 µm bacteriostatic polycarbonate filter, ad libitum (except during designated procedures)
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20 (the relative humidity was slightly higher than 70% on some occasions)
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

UNLABELLED TEST ITEM: administered directly to the animals

LABELLED TEST ITEM: added to the unlabelled test item to achieve a target radioactivity level of 10 µCi/animal of 14C-test article at a target dose of 500 mg/kg bw (dose volume: 0.5 mL/kg) and 5000 mg/kg bw (dose volume: 5 mL/kg)

Duration and frequency of treatment / exposure:

UNLABELLED TEST ITEM:
test group 2 and 3: 12 days, once daily
test group 4: 8 days, once daily

LABELLED TEST ITEM:
test group 2, 3 and 4: single application on day 6

Remarks:

Doses / Concentrations:
500, 5000 mg/kg bw day (0.5 or 5 mL/kg bw, respectively)

No. of animals per sex per dose / concentration:

1 (control), 24 (group 2 and 3), 5 (group 4)

Control animals:

yes, concurrent no treatment

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
TEST ANIMALS FROM GROUP 2 AND 3
- Tissues and body fluids sampled: blood, adipose tissue (perirenal), gastrointestinal tract and contents, kidneys, adrenals, liver and thymus
- Time and frequency of sampling: 1, 3, 6, 12, 24, 48, 72 and 168 h post labelled dose exposure
- Other: number of analysed animals/time point: 3
Remaining carcasses were collected from animals killed at 12, 24, 48 and 72 h only. All other remaining carcasses were discarded.


METABOLITE CHARACTERISATION STUDIES (test animals from group 4):
- Tissues and body fluids sampled: urine, faeces, cage washes (after 72 h post labelled dose exposure)
- Time and frequency of sampling: 6, 12, 24, 48 and 72 h post labelled dose exposure
- From how many animals: 5
- Other:
Expired 14CO2 was collected by drawing the cage air through a single collection tower (containing ca. 300 mL 4N KOH, target flow rate: 500-600 mL/min). Moisture and CO2 were removed from the air drawn through the cage by columns of anhydrous calcium chloride (Drierite) and Ascarite, respectively.
- Time and frequency of sampling: 3, 6, 12, 24, 48 and 72 h post radiolabelled dose.

Statistics:

Group mean values and standard errors were calculated from the examined parameters.

Details on absorption:

For groups 2 and 3, the highest radioactivity concentration in the tissues analyzed was observed at 1 h post treatment in the gastrointestinal tract and contents and then decreased over the 168 h period post dose. From the 48 h time point, the liver, the kidneys and adrenals and the thymus showed higher radioactivity concentrations than the gastrointestinal tract and contents in both groups. Thus, absorption of the test arcticle in the systemic circulation is present.
The radioactivity concentrations in adipose tissue (perirenal fat) increased from the 24 h time point throughout the study in group 2 and from the 12 h time point to the 72 h time point in group 3.

Details on distribution in tissues:

RADIOACTIVITY IN BLOOD AND PLASMA
500 mg/kg bw: blood and plasma radioactivity concentrations revealed their maximum 1 h post administration and decreased rapidly over the course of the study (1h: 106.1 µg eq/mL and 138.1 µg eq/mL; 168h: 15.2 µg eq/mL and 5.3 µg eq/mL for blood and plasma levels, respectively)
5000 mg/kg bw: blood and plasma radioactivity concentrations reached their maximum concentrations 6 h post treatment and decreased slowly (1h: 253.4 µg eq/mL and 300.7 µg eq/mL; 6h: 434.0 µg eq/mL and 672.4 µg eq/mL; 168h: 118.6 µg eq/mL and 40.5 µg eq/mL for blood and plasma levels, respectively)
Blood to plasma ratios in group 2 and group 3 animals were similar and lower than 1 (0.6 to 0.8) up to 24 h post administration indicating that little radioactivity was associated with blood cells. Further, less than 1.5% of the dose was found in the blood for animals of both test groups.

RADIOACTIVITY IN TISSUES AND GASTROINTESTINAL TRACT
low- and high-dose group: the highest radioactivity concentration in the tissues analysed was observed at 1 h post treatment in the gastrointestinal tract (approx. 2922 µg eq/g and 44050 µg eq/g for group 2 and group 3, respectively). These levels decreased over the course of the study to 12 µg eq/g and 122 µg eq/g for group 2 and 3 animals, respectively, representing both excretion of the test article from the gastrointestinal tract and absorption of the test article into the systemic circulation.
From the 48 h time point, the liver, the kidneys, adrenals and the thymus showed higher radioactivity concentrations than the gastrointestinal tract in both groups. For group 2 animals, the radioactivity concentrations in these tissues gradually declined over the 168 h period post-dose. For group 3 animals, the kidneys and adrenals, thymus and liver reached maximum concentrations at the 6, 12 and 24 h time point, respectively, and then declined for the rest of the observation period. The radioactivity concentrations in adipose tissue (perirenal fat) increased from the 24 h time point throughout the study in group 2 and from the 12 h time point to the 72 h time point in group 3. The radioactivity concentrations in adipose tissue (perirenal fat) then decreased until the 168 h time point for group 3 animals (at this time point, the radioactivity concentration in adipose tissue (perirenal fat) for 2 animals from group 3 could not be reported since the values measured were inconsistent and could not be reproduced). The highest tissue to plasma ratio was observed at 1 h post treatment in the gastrointestinal tract and contents at approximately 23 and 152 for group 2 and 3 animals, respectively. Due to excretion of the test article from the gastrointestinal tract, this ratio decreased rapidly over the course of the study to 2.4 for both groups at 168 h post dose. For kidneys and adrenals, the tissue to plasma ratio decreased from the 3 h time point to the 12 h time point in both groups. For most other tissues in both groups, the tissue to plasma ratios increased during the course of the study indicating a faster clearance from plasma than tissues. The lowest tissue to plasma ratios were observed in the adipose tissue (perirenal fat) of both groups.
The highest percentage of dose in tissues was observed in the gastrointestinal tract and contents at 1 h post dose for group 2 at approximately 50% and at 3 h post dose for group 3 at approximately 81% as expected since the test article was administered by gavage. Due to excretion and absorption of the test article, the percent of dose in the gastrointestinal tract decreased rapidly over the course of the study to 0.2% and 0.3% for group 2 and 3 animals, respectively, at 168 h post dose. The percent of dose in the carcasses was similar and constant for both groups at the selected time points (12 h, 24 h, 48 h and 72 h post administration) indicating that the test article may have been distributed in other tissues than the ones selected for analyses. Group 2 values ranged from 8.3% to 4.9% and group 3 values ranged from 7.2% to 5.4%. For group 2, the percentage of dose in the liver was 2.5% at 1 h post dose and decreased throughout the study to 0.2%. The percentage of dose in all other tissues for both groups was less than 1.5%. The lowest percentage of dose was found in the adipose tissue (less than 0.018%).

Details on excretion:

CLEARANCE FROM BLOOD vs PLASMA:
The blood to plasma ratio in test group 2 and 3 increased over the study period starting 48 h after substance administration. Thus, a faster clearance from the plasma than from blood is indicated.

CLEARANCE FROM TISSUE vs PLASMA:
For most tissues, the tissue to plasma ratios increased during the course of the study indicating a faster clearance from plasma than tissues.

EXCRETION AND MASS BALANCE
5000 mg/kg bw (group 4): the mean total recovery of radioactivity in the excreta of the 72 h period post dose was 108.5% of the dose (urine, 6.5%; feces, 24.6%; CO2, 77%; and cage wash, 0.5%). Most of the recovered radioactivity (97.5%) was excreted by 24 h post dose. The data demonstrated that the greatest amount of radioactivity was eliminated via the expired air, with some excretion via the feces. Little radioactivity was eliminated via the urine.
The mean mass balance of radioactivity at 72 h post-dose was 115% (ranging from 112% to 118%) with approximately 6.7% in the remaining carcass. The recovery was considered good.

Toxicokinetic parameters:

Cmax: 500 mg/kg bw: 106 µg eq/g (blood); 138 µg eq/g (plasma); 2922 µg eq/g (gastrointestinal tract); 338 µg eq/g (kidneys and adrenals); 332 µg eq/g (liver)

Toxicokinetic parameters:

Cmax: 5000 mg/kg bw: 434 µg eq/g (blood); 672 µg eq/g (plasma); 44 050 µg eq/g (gastrointestinal tract); 1533 µg eq/g (kidneys and adrenals); 1691 µg eq/g (liver)

Toxicokinetic parameters:

other: 500 mg/kg bw: tmax (blood and plasma): 1 h post administration; tmax (gastrointestinal tract): 1 h, tmax (tissue): 3 h; tmax (adipose tissue): 12 h

Toxicokinetic parameters:

other: 5000 mg/kg bw: tmax (blood and plasma): 6 h post administration; tmax (gastrointestinal tract): 1 h; tmax (adipose tissue): 72 h

Toxicokinetic parameters:

other: AUC0-tlast: 500 mg/kg bw: 4506 µg eq.h/g (blood value); 4384 µg eq.h/g (plasma value); 15 594 µg eq.h/g (gastrointestinal tract); 13 449 µg eq.h/g (kidneys and adrenals); 13 234 µg eq.h/g (liver)

Toxicokinetic parameters:

other: AUC0-tlast: 5000 mg/kg bw: 34 392 µg eq.h/g (blood value); 33 799 µg eq.h/g (plasma value); 515 329 µg eq.h/g (gastrointestinal tract); 116 002 µg eq.h/g (kidneys and adrenals); 129 372 µg eq.h/g (liver)

Toxicokinetic parameters:

other: elimination rate constant: 500 mg/kg bw: 0.0125/h (plasma); 0.00928 - 0.0162/h (tissues)

Toxicokinetic parameters:

other: elimination rate constant: 5000 mg/kg bw: 0.0134/h (plasma); 0.0112/h (gastrointestinal tract)

Toxicokinetic parameters:

half-life 1st: 500 mg/kg bw: 55.6 h (plasma); 43 - 75 h (tissues)

Toxicokinetic parameters:

half-life 1st: 5000 mg/kg bw: 51.9 h (plasma); 61.6 - 70.4 (tissues)

Toxicokinetic parameters:

other: AUC0-inf: 500 mg/kg bw: 4812 µg eq.h/g

Toxicokinetic parameters:

other: AUC0-inf: 5000 mg/kg bw: 36 833 µg eq.h/g (blood/plasma); 526 183 µg eq.h/g (gastrointestinal tract)

Metabolites identified:

not measured

ACTUAL DOSE RECEIVED

test group 2: 516 mg/kg bw

test group 3: 5015 mg/kg bw

test group 4: 5063 mg/kg bw

The mean 14C-radioactivity administered was:

test group 2: 8.45 µCi/animal

test group 3: 6.52 µCi/animal

test group 4: 5.93 µCi/animal

The slightly lower than targeted levels (10 µCi/animal) administered to animals was considered not to impact the outcome of the study since rats were dosed similar amounts of labelled and unlabelled test articles per kg of body weight.

CLINICAL OBSERVATIONS

No treatment related clinical signs were observed in animals prior to or subsequent to treatment with the test item.

LIPID ANALYSES

The recovery from the extraction of the gastrointestinal tract and contents, the liver and the blood was less than 50%. Thus, the radioactivity was associated mainly with water-soluble material. Additionally, the recovery in the blood was lower than 10%. One animal in group 3 showed a recovery of 60.7% in the gastrointestinal tract and contents at 1 hour post-dose. For the perirenal fat, the radioactivity was associated mainly with fat-soluble material.

Thin layer chromatography of organic extracts 1 hour after dosing demonstrated an association of the radioactivity in the gastrointestinal tract and contents with free fatty acids for the low and high-dose group. Further, 12 and 24 hours post-dosing, the radioactivity in the gastrointestinal tract and contents for the high-dose group was mostly associated with diacylglycerides.

Liver extracts revealed an association of the radioactivity with cholesterol for group 3 animals in samples isolated 6 h after administration. However, the exact nature of the main component retained on the origin could not be determined although it was determined for the standards that phospholipids also had an Rf value of 0. In one animal of the low dose group, more than 70% of the radioactivity in the blood extract was cholesterol whereas another animal of the same dose group showed background levels of the radioactivity in the blood extracts. For perirenal fat, the radioactivity in the extracts was also at background level.

Conclusions:

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

Additional information

Justification for grouping of substances and read-across

The Glycerides category covers aliphatic (fatty) acid esters of glycerol. The category contains both well-defined and UVCB substances with aliphatic acid carbon chain lengths of C2 (acetate) and C7-C22, which are mostly linear saturated and even numbered. Some of the substances in the category contain unsaturated fatty acids (e.g. oleic acid in 2,3-dihydroxypropyl oleate, CAS 111-03-5 or general fatty acids C16-22 (even) unsaturated in Glycerides, C14-18 and C16-22-unsatd., mono- and di-, CAS 91744-43-7). Some category members contain branched fatty acids. Branching is mostly methyl groups (e.g. isooctadecanoic acid, monoester with glycerol, CAS 66085-00-5 or 1,2,3-propanetriyl triisooctadecanoate, CAS 26942-95-0). In one category member the branching cannot be precisely located (Glycerides, C16-18 and C18-unsatd., branched and linear mono-, di- and tri, ELINCS 460-300-6). Hydroxylated fatty acids are present in three substances (Castor oil, CAS 8001-79-4; castor oil hydrogenated, CAS 8001-78-3 and 2,3-dihydroxypropyl 12-hydroxyoctadecanoate, CAS 6284-43-1). Hydroxylation occurs on C12 of stearic acid in all these substances. Acetylated chains are present in the last part of the category, comprising fatty acids from C8 to C18 (even) and also C18 unsaturated, additionally a C18 acetylated fatty acid is present with the acetic acid located in C12 position (e.g. Glycerides, castor oil mono-, hydrogenated acetates / 12-acetoxy-octadecanoic acid, 2,3-diacetoxy, CAS 736150-63-3). All glycerides build mono-, di- and tri-esters in variable proportions.

Fatty acid esters are generally produced by chemical reaction of an alcohol (e.g. glycerol) with an organic acid (e.g. acetic, stearic or oleic acid) in the presence of an acid catalyst (Radzi et al., 2005). The esterification reaction is started by the transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The carboxylic acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to the carbonyl carbon of the acid. An intermediate product is formed. This intermediate product loses a water molecule and proton to give an ester (Liu et al., 2006; Lilja et al., 2005; Gubicza et al., 2000; Zhao, 2000). Mono-, di- and tri-esters are the final products of esterification with glycerol.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby substances may be considered as a category provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity, the substances listed below are allocated to the category of Glycerides.

Glycerides category members include

CAS	EC name	Molecular weight (range in case of UVCBs)	Fatty acids chain length	Degree of esterification	Molecular formula
26402-26-6	Octanoic acid, monoester with glycerol	218.29	C8	Mono	C11H22O4
142-18-7	2,3-dihydroxypropyl laurate	274.40	C12	Mono	C15H30O4
25496-72-4	Oleic acid, monoester with glycerol	356.54	C18:1	Mono	C21H40O4
111-03-5	2,3-dihydroxypropyl oleate	356.54	C18:1	Mono	C21H40O4
66085-00-5	Isooctadecanoic acid, monoester with glycerol	358.55	C18iso	Mono	C21H42O4
6284-43-1	2,3-dihydroxypropyl 12-hydroxyoctadecanoate	374.56	C18OH	Mono	C21H42O5
620-67-7	Propane-1,2,3-triyl trisheptanoate	428.60	C7	Tri	C24H44O6
538-23-8	Glycerol trioctanoate	470.68	C8	Tri	C27H50O6
538-24-9	Glycerol trilaurate	639.00	C12	Tri	C39H74O6
122-32-7	1,2,3-propanetriyl trioleate	885.43	C18:1	Tri	C57H104O6
555-43-1	Glycerol tristearate	891.48	C18	Tri	C57H110O6
26942-95-0	1,2,3-propanetriyl triisooctadecanoate	891.48	C18iso	Tri	C57H110O6
91052-47-0	Glycerides, C16-18 mono-	330.51 - 358.56	C16, C18	Mono	C19H38O4; C21H42O4
91744-09-1	Glycerides, C16-18 and C18-unsatd. mono-	330.51 - 358.56	C16, C18; C18uns.	Mono	C19H38O4; C21H42O4; C21H40O4
85536-07-8	Glycerides, C8-10 mono- and di-	218.29 - 400.60	C8, C10	Mono and di	C11H22O4; C13H26O4; C19H36O5; C23H44O5
91052-49-2	Glycerides, C12-18 mono- and di-	274.40 - 625.04	C12, C14, C16, C18	Mono and di	C15H30O4; C21H42O4; C27H52O5; C39H76O5
67701-33-1	Glycerides, C14-18 mono- and di-	302.45 - 625.02	C14, C16, C18	Mono and di	C17H34O4; C21H42O4; C31H60O5; C39H76O5
67784-87-6	Glycerides, palm-oil mono- and di-, hydrogenated	302.45 - 625.02	C14, C16, C18	Mono and di	C17H34O4; C21H42O4; C31H60O5; C39H76O5
91845-19-1	Glycerides, C16-18 and C18-hydroxy mono- and di-	330.51 - 657.02	C16, C18 C18OH	Mono and di	C19H38O4; C21H42O4; C35H68O5; C39H76O5; C21H42O5; C39H76O7
97358-80-0	Isooctadecanoic acid, mono- and diesters with glycerol	358.57 - 625.02	C18iso	Mono and di	C21H42O4; C39H76O5
91744-13-7	Glycerides, C14-18 and C16-22-unsatd. mono- and di-	302.45 - 733.20	C14, C16, C18, C16, C18 and C22uns.	Mono and di	C17H34O4; C21H42O4; C19H36O4; C25H48O4; C31H60O5; C39H76O5; C35H64O5; C47H88O5
31566-31-1	stearic acid, monoester with glycerol	325.03 - 330.51	C16, C18	Mono and di	C19H38O4; C21H42O4; C35H68O5, C39H76O5
85251-77-0	Glycerides, C16-18 mono- and di-	330.51 - 625.03	C16, C18	Mono and di	C19H38O4; C21H42O4; C35H68O5; C39H76O5
91744-32-0	Glycerides, C8-10 mono-, di- and tri-	218.29 - 554.84	C8, C10	Mono, di and tri	C11H22O4; C13H26O4; C19H36O5; C23H44O5; C27H50O6; C33H62O6
91052-28-7	Glycerides, C14-18 and C16-18-unsatd. mono-, di- and tri-	302.46 - 885.46	C14, C16, C18, C16:1, C18:1, C18:2, C18:3	Mono, di and tri	C17H34O4; C21H42O4; C19H36O4; C21H40O4; C31H60O5; C39H76O5; C35H64O5; C39H72O5; C45H86O6; C57H110O6; C51H92O6; C57H104O6
91052-54-9	Glycerides, C16-18 mono-, di- and tri-	330.50 - 891.48	C16, C18	Mono, di and tri	C19H38O4; C21H42O4; C35H68O5; C39H76O5; C51H98O6; C57H110O6
91744-20-6	Glycerides, C16-18 and C18-unsatd. mono-, di and tri-	330.51 - 891.50	C16, C18, C18uns.	Mono, di and tri	C19H38O4; C35H68O5; C51H98O6; C21H40O4; C39H72O5; C57H104O6
no CAS	ELINCS 460-300-6: Glycerides, C16-C18 and C18-unsaturated, branched and linear mono-, di- and tri-	330.51 - 891.50	C16, C18, C18uns., branched and linear	Mono, di and tri	C19H38O4; C35H68O5; C51H98O6; C21H40O4; C39H72O5; C57H104O6
97722-02-6	Glycerides, tall-oil mono-, di-, and tri-	356.54 - 885.43	C16, C18, C20,C18uns.	Mono, di and tri	C21H40O4; C39H72O5; C57H104O6
77538-19-3	Docosanoic acid, ester with 1,2,3-propanetriol	414.66 - 1059.80	C22	Mono, di and tri	C25H50O4; C47H92O5; C69H134O6
91744-28-4	Glycerides, C12-18 di- and tri-	456.70 - 891.50	C12, C14, C16, C18	Di and tri	C27H52O5; C39H76O5; C39H74O6; C57H110O6
68606-18-8	Glycerides, mixed coco, decanoyl and octanoyl	470.69 - 807.32	C8, C10, C12, C14, C16	Di and tri	C27H50O6; C33H62O6; C39H74O6; C45H86O6; C51H98O6
65381-09-1	Decanoic acid, ester with 1,2,3-propanetriol octanoate	470.69 - 554.85	C8, C10	Tri	C27H50O6; C33H62O6
73398-61-5	Glycerides, mixed decanoyl and octanoyl	470.69 - 554.85	C8, C10	Tri	C27H50O6; C33H62O6
85536-06-7	Glycerides, C8-18	470.68 - 891.48	C8, C10, C12, C14, C16, C18	Tri	C27H50O6; C57H110O6
67701-26-2	Glycerides, C12-18	639.01 - 891.48	C12, C14, C16, C18	Tri	C39H74O6; C57H110O6
67701-30-8	Glycerides, C16-18 and C18-unsatd.	807.32 - 891.48	C16, C18; C18uns.	Tri	C21H40O4; C39H72O5; C57H104O6
8001-79-4	Castor oil	933.43	C18:1(OH)	Tri	C57H104O9
8001-78-3	Castor oil, hydrogenated	939.48	C18OH	Tri	C57H110O9
97593-30-1	Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates	330.42 - 442.63	C2; C10	Tri (FA mono, diacetate)	C17H30O6; C25H46O6
97593-30-1	Glycerides, C8-21 and C8-21-unsatd. mono- and di-, acetates	358.47 - 498.74	C2; C12	Tri (FA mono, diacetate)	C19H34O6; C29H54O6
93572-32-8	Glycerides, palm-oil mono-, hydrogenated, acetates	372.54 - 400.59	C2; C16	Tri (FA mono, diacetate)	C21H40O5; C23H44O5
91052-13-0	Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates	302.36 - 582.91	C2; C8, C10, C12, C14, C16, C18, C18uns.	Tri	C15H26O6; C19H34O6; C21H38O6; C25H46O6
736150-63-3	Glycerides, castor-oil-mono, hydrogenated, acetates (main component: 12-acetoxy-octadecanoic acid (2,3-diacetoxy)propyl ester [CAS 330198-91-9])	500.67	C2; C18Ac	Tri (FA mono, diacetate)	C27H48O8
no CAS (c, d)	Short-, medium- and long-chain triglycerides (SCT, MCT, LCT)	-	C2-C18 (even numbered), C18uns.	Tri	-
no CAS (c, d)	mixture of mono-, di-, and triglycerides of lauric acid	274.40 - 639.00	C12	Mono, di and tri	C15H30O4; C27H52O5; C39H74O6
no CAS (c, d)	Modified triglyceride. Main components: 1,3-dioleoyl 2-palmitoyl triacylglycerol and 1,2-dipalmitoyl 3-oleoyl triacylglycerol	833.36 - 859.39	C16, C18, C18uns.	Tri	C53H100O6; C55H102O6
56-81-5 (c)	Glycerol	92.09	--	--	C3H8O3
111-14-8 (c)	Heptanoic acid	130.18	C7	--	C7H14O2
112-85-6 (c)	Docosanoic acid	340.58	C22	--	C22H44O2

(c) Surrogate substances are either chemicals forming part of a related category of structurally similar fatty acid esters or precursors/breakdown products of category members (i.e. alcohol and fatty acid moieties). Available data on these substances are used for assessment of (eco )toxicological properties by read-across on the same basis of structural similarity and/or mechanistic reasoning as described below for the present category.

(d) Assessment of toxicological properties is conducted also taking into account available data on mixtures of synthetic and/or naturally occurring glycerides (e.g. vegetable oils), which cannot be identified by a (single) CAS/EC number. The test materials short-, medium- and long-chain triglycerides (SCT, MCT, LCT) and their combinations (e.g. MLCT, SALATRIM – a SLCT) comprise triesters of glycerol with fatty acid chain lengths of C2 and C4 (short-chain), C8 and C10 (medium-chain) and C18 saturated/unsaturated (long-chain). The substance “mixture of mono-, di-, and triglycerides of lauric acid” comprises mono-, di and triesters of glycerol with dodecanoic acid (C12). The substance “Modified triglyceride” contains main components: 1,3-dioleoyl 2-palmitoyl triacylglycerol and 1,2-dipalmitoyl 3-oleoyl triacylglycerol, comprising triesters of glycerol with hexadecanoic (C16) and (9Z)-Octadec-9-enoic acid (C18:1). Available data on identity and composition of the individual test material for a given study is provided in the technical dossier.

 

Grouping of substances into this category is based on:

(1) common functional groups: all members of the Glycerides category are esters of a tri-functional alcohol (glycerol) with one or more carboxylic (fatty) acid(s) chain(s). The alcohol moiety (glycerol) is common to all category members. The fatty acid moiety comprises carbon chain lengths of C2 (acetate) and from C7-C22 (uneven/even-numbered) and includes mainly linear saturated alkyl chains, but also unsaturated, branched, hydroxylated and acetylated chains bound to the alcohol, resulting in mono-, di-, and tri-esters; and

(2) common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals: all members of the Glycerides category result from esterification of glycerol with the respective fatty acid(s). Esterification is, in principle, a reversible reaction (hydrolysis). Thus, the glycerol and fatty acid moieties are simultaneously precursors and breakdown products of Glycerides. For the purpose of grouping of substances, enzymatic hydrolysis in the gastrointestinal tract and/or liver is identified as the biological process, by which the breakdown of Glycerides result in structurally similar chemicals. Furthermore, hydrolysis represents the first chemical step in the absorption, distribution, metabolism and excretion pathways anticipated to be similarly followed by all Glycerides (CIR, 1984, 2004, 2007; Elder, 1990, 1982, 1986; FDA, 1975; Johnson, 2001; Lehninger, 1998; NTP, 1994; Stryer, 1996; WHO, 1967, 1974, 1975, 1979, 2001). Hydrolysis is catalysed by a class of enzymes known as lipases, a subgroup of carboxylesterases. In general, Glycerides are enzymatically hydrolysed in the small intestine to glycerol and corresponding carboxylic acid(s), and in the case of di- and triglycerides also to monoglycerides (with the ester bond at the sn-2 position). Following hydrolysis, glycerol is readily absorbed through the gastrointestinal tract and can be re-esterified to form endogenous glycerides or be metabolised to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, which can be incorporated in the standard metabolic pathways of glycolysis and gluconeogenesis. Being a polar molecule, glycerol can also be readily excreted in the urine. Fatty acids are likewise readily absorbed by the intestinal mucosa and distribute systemically. Fatty acids are a source of energy. They are either re-esterified into triacylglycerols and stored in adipose tissue, or enzymatically degraded for energy primarily via β-oxidation. Alternative oxidation pathways (alpha- and omega-oxidation) are available and are relevant for degradation of branched fatty acids. Unsaturated fatty acids require additional isomerization prior to enter the β-oxidation cycle. Acetate, resulting from hydrolysis of acetylated Glycerides, is readily absorbed and feeds naturally into physiological pathways of the body and can be utilized in oxidative metabolism or in anabolic syntheses; and

(3) constant pattern in the changing of the potency of the properties across the category: the available data show similarities and trends within the category in regard to physicochemical, environmental fate, ecotoxicological and toxicological properties. For those individual endpoints showing a trend, the pattern in the changing of potency is clearly and expectedly related to the length of the fatty acid chains and the degree of substitution of glycerol (mono-, di- or triester).

a) Physicochemical properties:

The physico-chemical properties of the category members are similar or follow a regular pattern over the category. The patterns observed depend on the fatty acid chain length and the degree of esterification (mono-, di- or triester).

The molecular weight of the category members (glycerol esters) ranges from 218.29 to 1059.80 g/mol. The physical state is related to the chain length of the fatty acid moiety, the degree of saturation and the number of ester bonds. Thus, monoesters of short- and long-chain fatty acids (C8-C12) as well as unsaturated (C18:1) fatty acids and C18OH are solids, whereas monoesters of branched fatty acids (C18iso) are liquids. Triesters of shorter-chain fatty acids (C8-12) as well as unsaturated (C18:1) and branched longer-chain acids (C18iso) are liquids. The physical state of mixtures of mono-, di- and tri-esters depends on the amount of different esters. Mono-, di- and triesters of shorter-chain fatty acids are liquid (C8-12), mono-, di- and triesters of longer-chain fatty acids are solids (C14-18, C18OH and also C18iso). The turning point of this property seems to be fatty acids C12. In addition, mono- and diesters with a certain amount of unsaturated acids are liquids. Following the described pattern the UVCB triesters of shorter-chain fatty acids (C8-14) and unsaturated fatty acids (C18:1 and C18:1OH) are liquids. For the glycerides with acetic acid (mainly monoester of fatty acids and diester of acetic acid) the turning point seems to be the fatty acid chain length C14/C16. Below this point the substances are liquid, above this point category members are solid.

Also the boiling points are following a pattern: Increasing molecular weight results in increasing boiling temperatures. For a molecular weight of below 300 g/mol the boiling point is around 170 °C (C12 monoester), between a molecular weight of 350 to 480 g/mol the boiling point is between 230-300 °C. Above 300 g/moles the decomposition of the substances is probable. Also the acetate esters have boiling points >300 °C. According to Blake et al. (J. Chem. Eng. Data, 1961, 6, 87-98), esters of long chain acids with β‑hydrogen atoms in the alcohol moiety (i.e. alcohols with C3, e.g. propanol) decompose in the range between 262 and 283 °C. Since for longer chains the boiling temperature is higher, esters of fatty acids esterified with alcohols ≥ C3 and having a molecular weight exceeding 300 amu have a boiling point >300 °C and decompose before boiling.

All category members are non-volatile with a vapour pressure <0.01 Pa at temperature of 20 °C, mainly based on (Q)SAR calculation.

The n-octanol/water partition coefficient increases with increasing chain length and increasing degree of esterification (e.g. C8 monoester: 1.71; C7 triester: 8.86; C22 triester >15). A positive correlation with the overall number of CH2 units is observed.

The water solubility decreases accordingly with increasing chain length or increasing overall number of CH2 units (20-60 mg/L for C8 monoester to <0.05 mg/L for C7 triester; <4 mg/L for C18:1 monoester to <0.05 mg/L for C18iso monoester). The cut-off value for water solubility below 1 mg/L seems to be the C16 to C18 monoester. For higher degree of esterification (di and triesters) other limits are applicable: a C12 diester at least has a water solubility of below 1 mg/L, the C7 triester has solubility well below 1 mg/L. The water solubility depends on the method used for testing and for analysis of test item. Testing by GC-MS is more selective than testing by TOC/DOC method, GC-MS results are therefore lower than results obtained by TOC. Nevertheless a correlation between increasing molecular weight and decreasing water solubility can be found.

b) Environmental fate and ecotoxicological properties:

The members of the Glycerides category are readily biodegradable and show low bioaccumulation potential in biota. Hydrolysis is not a relevant degradation pathway for these substances, due to their ready biodegradability and estimated half-lives in water > 250 days at pH 7 and 25 days at pH 8 (HYDROWIN v2.00).The majority of the Glycerides category members have log Koc values > 3, indicating potential for adsorption to solid organic particles. Therefore, the main compartments for environmental distribution of these substances are expected to be soil and sediment, with the exception of 2,3-dihydroxypropyl laurate (CAS 142-18-7), for which a log Koc < 3 is reported. Therefore, this substance will be most likely available in the water phase. Nevertheless, all substances are readily biodegradable, indicating that persistency in the environment is not expected. The volatilization potential of the Glycerides category members is negligible, based on vapour pressure values ranging from < 0.0001 Pa to < 5 Pa at 20°C. Nevertheless, if released into the atmosphere, these substances are expected to be rapidly photodegraded in view of their estimated half-lives in air, ranging from 1.5 to 20.7 hours (AOPWIN 1.92 program). Based on the above information, accumulation in air, subsequent transportation through the atmosphere and deposition into other environmental compartments is not anticipated. Regarding aquatic toxicity, acute and chronic values obtained in tests conducted on fish, invertebrates, algae and microorganisms showed no adverse effects in the range of the water solubility of the substances (or the highest attainable solubility in aqueous medium), with the exception of Glycerides, palm-oil mono-, hydrogenated, acetates (CAS 93572-32-8). Even though it cannot be excluded that for this substance the observed effects are due to physical interference with undissolved test material (particulate material observed in test solutions), the NOEC value of the algae test is < 1 mg/L (0.565 mg/L) and within the water solubility range of the substance (1.3-7.4 mg/L). Therefore, a conservative approach is applied and the substance classified as environmental hazard Chronic category 3, according to Regulation (EC) No. 1272/2008. Based on the available data, no toxicity to aquatic microorganisms, sediment and terrestrial organisms is to be expected for the substances of the Glycerides category.

c) Toxicological properties:

The available data shows that the category of Glycerides is characterised by a lack of change of the potency of toxicological properties. No human health hazard is identified. Thus, all available studies consistently show that Glycerides are not acutely toxic via the oral, dermal and inhalation routes. The available animal and human studies indicate that Glycerides are not skin or eye irritating and not skin sensitising. All available in vitro and in vivo genetic toxicity studies are negative for the induction gene mutations in bacteria and mammalian cells and of chromosome aberrations or micronuclei in mammalian cells. No adverse effects were observed up to, including and even well above the limit dose of 1000 mg/kg bw/day in the available short- and long-term toxicity studies via the oral route. Likewise, no reproductive toxicity effects were observed in any of the available studies.

 

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate, environmental and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

 

Basic toxicokinetics

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2012), assessment of the toxicokinetic behaviour of the substance is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physicochemical and toxicological properties according to the relevant Guidance (ECHA, 2012) and taking into account further available information from members of the Glycerides category. There are no studies available in which the toxicokinetic behaviour of the substance has been investigated.

The substance Octanoic acid, monoester with glycerol (CAS 26402-26-6) is mainly a monoester of glycerol and linear, even-numbered saturated fatty acids with C8 (octanoic acid) chain lengths.

Octanoic acid, monoester with glycerol has a molecular weight of 218 g/mol. The substance is a white solid with characteristic odour at 20 °C with a melting point of 38 °C at normal pressure, a water solubility of 330 g/L and vapour pressure of < 0.01 Pa at 20 °C was determined for the substance.

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2012).

Oral

In general, molecular weights below 500 and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (> 1 mg/L). Lipophilic compounds may be taken up by micellar solubilisation by bile salts, but this mechanism may be of particular importance for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) which would otherwise be poorly absorbed (Aungst and Chen, 1986; ECHA, 2012).

The physicochemical characteristics (log Pow and water solubility) of the substance and the molecular weight distribution are in a range suggestive of low absorption from the gastrointestinal tract subsequent to oral ingestion.

The potential of a substance to be absorbed in the (GI) tract may be influenced by chemical changes taking place in GI fluids as a result of metabolism by GI flora, by enzymes released into the GI tract or by hydrolysis. These changes will alter the physicochemical characteristics of the substance and hence predictions based upon the physico-chemical characteristics of the parent substance may no longer apply (ECHA, 2012).

It is well-accepted knowledge that triglycerides (e.g. from dietary fat) undergo hydrolysis by lipases (a class of ubiquitous carboxylesterases) prior to absorption; and there is sufficient evidence to assume that all of the mono-, di- and triglycerides contemplated within the category of Glycerides will likewise undergo enzymatic hydrolysis in the GI tract as the first step in their absorption, distribution, metabolism and excretion (ADME) pathways as summarised below.

In the gastrointestinal tract, gastric and intestinal (pancreatic) lipase activities are the most important. Triglycerides are hydrolysed by gastric and pancreatic lipases with high specificity for the sn1- and sn3-positions. For the remaining monoester at the sn2-position (2-monoacylglycerol), there is evidence that it can either be absorbed as such by the intestinal mucosa or isomerize to 1-monoacylglycerol, which can then be hydrolysed. The rate of hydrolysis by gastric and intestinal lipases depends on the carbon chain length of the fatty acid moiety. Thus, triesters of short-chain fatty acids are hydrolysed more rapidly and to a larger extent than triesters of long-chain fatty acids. (Barry et al., 1966; Cohen et al.,1971; Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1964, 1966, 1968; WHO, 1967, 1975). In a recent study conducted with the structurally related substance Glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3), rapid ester hydrolysis in intestinal fluid simulant was confirmed (Jensen, 2002).

Octanoic acid, monoester with glycerol is therefore anticipated to be enzymatically hydrolysed to glycerol and saturated C8 (octanoic acid) fatty acids, respectively.

Following hydrolysis, the resulting products free glycerol, free fatty acids, and (in the case of di- and triglycerides) 2-monoacylglycerols are absorbed by the intestinal mucosa. Within the epithelial cells, triglycerides are reassembled, primarily by re-esterification of absorbed 2-monoacylglycerols. Thus, free glycerol is readily absorbed independently of the fatty acids and little of it is re-esterified. As for hydrolysis, the absorption rate of free fatty acids is chain length-dependent. The absorption of short-chain carboxylic acids can therefore begin already in the stomach. In general, for intestinal absorption short-chain or unsaturated carboxylic acids are more readily absorbed than long-chain, saturated fatty acids. However, the absorption of saturated long-chain fatty acids is increased if they are esterified at the sn2-position of glycerol (Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1962, 1964). Recently a study was conducted with 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester, serving as surrogate for the substance Glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3) to investigate the pharmacokinetics, tissue distribution, excretion and mass balance of radioactivity in rats after a single oral dose of the test material (St-Pierre, 2004). The results of the study showed that the test material, specifically the fatty acid moiety, was readily absorbed from the gastrointestinal tract, systemically distributed and metabolised. Based on the reported data on mass balance of radioactivity, absorption was higher than 80%. A high rate of absorption was also demonstrated in a feeding study with soybean oil in rats, resulting in oral absorption of 95 -98% when administered at 17% of the diet (Nolen, 1972). Furthermore, for palmitic acid it was shown that absorption rate was depending on the form in which it was fed, i.e. absorption was greatest when palmitic acid was fed as β-palmitoyl diolein, and least when it was fed as the free acid (Mattson and Volpenhein, 1962).

In conclusion, based on the available information, the physicochemical properties and molecular weight of Octanoic acid, monoester with glycerol

suggest low oral absorption. However, the substance is anticipated to undergo enzymatic hydrolysis in the gastrointestinal tract and absorption of the ester hydrolysis products rather than the parent substance is likely. The absorption rate of the hydrolysis products is considered to be high.

Dermal

The dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Dermal uptake is anticipated to be low, if the water solubility is < 1 mg/L; low to moderate if it is between 1-100 mg/L; and moderate to high if it is between 100-10000 mg/L. Dermal uptake of substances with a water solubility > 10000 mg/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values in the range of 1 to 4 (values between 2 and 3 are optimal) are favourable for dermal absorption, in particular if water solubility is high. For substances with a log Pow above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Log Pow values above 6 reduce the uptake into the stratum corneum and decrease the rate of transfer from the stratum corneum to the epidermis, thus limiting dermal absorption (ECHA, 2012).

The physicochemical properties (log Pow and water solubility) of the substance and the molecular weight are in a range suggestive of low absorption through the skin.

If a substance shows skin irritating or corrosive properties, damage to the skin surface may enhance penetration. If the substance has been identified as a skin sensitizer then some uptake must have occurred although it may only have been a small fraction of the applied dose (ECHA, 2012).

Two studies are available investigating the skin irritation potential of Glycerides, mixed decanoyl and octanoyl, resulting in no skin reactions in rabbits following application of the substance (Jones, 1988; Klimmer, 1971). Furthermore, a skin sensitisation study in guinea pigs did not show any skin sensitisation reactions for the substance at a concentration of 4% in ethanol. The non-sensitising potential was further evidenced by a study with the structurally related substance Propane-1,2,3-triyl trisheptanoate (CAS 620-67-7), showing no skin sensitisation reactions in guinea pigs after challenge treatment (Mürmann, 1993). Based on studies with the substance itself and those of structural analogues,

Octanoic acid, monoester with glycerol

is not expected to cause any skin irritation and sensitisation reactions, which might enhanced penetration of the substance due to local skin damage and thus increase dermal absorption.

Overall, taking all available information into account, the dermal absorption potential is considered to be low.

Inhalation

Octanoic acid, monoester with glycerol is a liquid with very low vapour pressure of < 0.01 Pa at 20 °C, thus being of very low volatility. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapours, gases, or mists is not significant.

However, the substance may be available for respiratory absorption in the lung after inhalation of aerosols, if the substance is sprayed (e.g. as a formulated product). In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract (ECHA, 2012).

As for oral absorption, the molecular weight, log Pow and water solubility are suggestive of absorption across the respiratory tract epithelium either by micellar solubilisation.

Overall, systemic bioavailability is considered likely after inhalation of aerosols with aerodynamic diameters below 15 µm.

Distribution and Accumulation

Distribution of a compound within the body depends on the physicochemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration, particularly in fatty tissues (ECHA, 2012).

As discussed under oral absorption, mono-, di- and triesters of glycerol undergo enzymatic hydrolysis in the gastrointestinal tract prior to absorption. Therefore, assessment of distribution and accumulation of the hydrolysis products is considered more relevant.

Absorbed glycerol is readily distributed throughout the organism and can be re-esterified to form endogenous triglycerides, be metabolised and incorporated into physiological pathways or be excreted in the urine. After being absorbed, fatty acids are (re-)esterified along with other fatty acids into triglycerides and released in chylomicrons into the lymphatic system (Bergström, 1951). Fatty acids of carbon chain length ≤ 12 may be transported as the free acid bound to albumin directly to the liver via the portal vein, instead of being re-esterified. Chylomicrons are transported in the lymph to the thoracic duct and eventually to the venous system. Upon contact with the capillaries, enzymatic hydrolysis of chylomicron triacylglycerol fatty acids by lipoprotein lipase takes place. Most of the resulting fatty acids are taken up by adipose tissue and re-esterified into triglycerides for storage. Triacylglycerol fatty acids are likewise taken up by muscle and oxidized for energy or they are released into the systemic circulation and returned to the liver (IOM, 2005; Johnson, 1990; Johnson, 2001; Lehninger, 1998; NTP, 1994; Stryer, 1996; WHO, 2001; Matulka, 2009).

Stored fatty acids underlie a continuous turnover as they are permanently metabolised for energy and excreted as CO2. Bioaccumulation of fatty acids takes place, if their intake exceeds the caloric requirements of the organism.

In the study by St-Pierre (2004) with 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester (surrogate of Glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3), systemic distribution of the radiolabelled material was confirmed in rats. Radioactivity was detected in all tissues and organs sampled (adipose tissue, gastrointestinal tract and content, kidneys and adrenals, liver, thymus and the remaining carcass) with highest levels recovered in the gastrointestinal tract, liver and the remaining carcass. Due to excretion and absorption of the radiolabelled material, the radioactivity content in the gastrointestinal tract decreased rapidly over the course of the study (168 h). This was similar for the radioactivity recovered in liver, whereas the radioactivity found in the carcasses was nearly constant at the selected time points, indicating that the radiolabelled material may have been distributed to other tissues than the ones selected for analyses. Based on the results of this study, no bioaccumulation potential was observed for 12-acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester.

Metabolism

Glycerol can be metabolised to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, which can then be incorporated in the standard metabolic pathways of glycolysis and gluconeogenesis. Fatty acids are degraded by mitochondrial β-oxidation which takes place in the most animal tissues and uses an enzyme complex for a series of oxidation and hydration reactions resulting in the cleavage of acetate groups in form of acetyl CoA. The alkyl chain length is thus reduced by 2 carbon atoms in each β-oxidation cycle. The complete oxidation of unsaturated fatty acids such as oleic acid requires an additional isomerisation step. Alternative pathways for oxidation can be found in the liver (ω-oxidation) and the brain (α-oxidation). Thus iso-fatty acids such as isooctadecanoic acid have been found to be activated by acyl coenzyme A synthetase of rat liver homogenates and to be metabolised to a large extent by ω-oxidation. Each two-carbon unit resulting from β-oxidation enters the citric acid cycle as acetyl CoA, through which they are completely oxidized to CO2. Acetate, resulting from hydrolysis of acetylated Glycerides, is readily absorbed and feeds naturally into physiological pathways of the body and can be utilized in oxidative metabolism or in anabolic syntheses (CIR, 1983, 1987; IOM, 2005; Lehninger, 1998; Lippel, 1973; Stryer, 1996; WHO, 1967, 1974, 1975, 2001; Adolph, 1999).

Excretion

As far as Glycerides are not hydrolysed in the gastrointestinal tract, they are excreted in the faeces.

In general, the hydrolysis products glycerol and fatty acids are catabolised entirely by oxidative physiologic pathways ultimately leading to the production of carbon dioxide and water. Glycerol, being a polar molecule can readily be excreted in the urine. Small amounts of ketone bodies resulting from the oxidation of fatty acids are excreted via the urine (Lehninger, 1998; IOM, 2005; Stryer, 1996).

In rats given a single dose of 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester at 5000 mg/kg bw, the mean total recovery of radioactivity in the excreta of the 72 h period post-dose was 108.5% of the dose (urine, 6.5%; faeces, 24.5%; CO2, 77%; and cage wash, 0.5%). Most of the recovered radioactivity (97.5%) was excreted by 24 h post dose (St-Pierre, 2004). The results thus confirm that Glycerides are mainly excreted as CO2 in the expired air as a result of metabolism.