Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jul 2003 - 19 Jan 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Endpoint:
basic toxicokinetics
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
11 Jul 2003 - 19 Jan 2004
Reason / purpose for cross-reference:
read-across source
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
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting 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:
No bioaccumulation potential based on study results
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reason / purpose for cross-reference:
read-across source
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:
No bioaccumulation potential based on study results

Description of key information

The target substance Dub TGI 24 is expected to be hydrolysed within the gastrointestinal tract and the hydrolysis products are predicted to be readily absorbed via the oral and inhalation route, and partly absorbed via the dermal route. The ester bonds will be hydrolysed in the gastrointestinal tract and mucus membranes to the respective fatty acid and glycerol, which facilitates the absorption. The absorbed ester fraction will be hydrolysed mainly in the liver. The fatty acid will most likely be re-esterified to triglycerides after absorption and transported via chylomicrons; the absorbed glycerol is readily distributed throughout the organism and can be re-esterified to form endogenous triglycerides. The major metabolic pathway for linear and branched fatty acids is the beta-oxidation pathway for energy generation, while alternatives are the omega-pathway or direct conjugation to more polar products. The excretion will mainly be as COin expired air; with a smaller fraction excreted as conjugated molecules in the urine. Glycerol can likewise be metabolised and incorporated into physiological pathways. No bioaccumulation will take place, as excess triglycerides are stored and used as the energy need rises.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

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, 2014), an assessment of the toxicokinetic behaviour of the target substance Dub TGI 24 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 Chapter R.7c Guidance document (ECHA, 2014) and taking into account further available information from source substances. There are no studies available in which the toxicokinetic behaviour of Dub TGI 24 was investigated.

Dub TGI 24 is a UVCB substance. The substance is a liquid at 20 °C with a melting point of app. <-10 °C at normal pressure, estimated water solubility of < 0.05 mg/L at 25 °C and estimated vapour pressure of < 0.0001 Pa at 20 °C. The log Pow was estimated to be > 10 (refer to IUCLID section 4.7).

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, 2014).

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; this mechanism is important for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) as these would otherwise be poorly absorbed (Aungst and Chen, 1986; ECHA, 2014).

The lower end of the molecular weight range of the Dub TGI 24 constituents indicates absorption from the gastrointestinal tract following oral ingestion is possible. However, the partition coefficient (log Pow) and the upper end of the molecular weight range indicate poor absorption. It is unclear to what degree micellar solubilisation will affect the absorption rate of the substance, as it has low water solubility.

The available data on acute and repeated dose oral toxicity support a conclusion of no/low toxicity. When mice were administered a single dose of 2000 mg/kg bw Dub TGI 24, there was no mortality, no adverse clinical signs and no adverse effects on body weight (please refer to IUCLID section 7.2.1). No adverse effects were observed in two subacute repeated dose toxicity studies (Combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, according to OECD guideline 422, please refer to IUCLID section 7.5.1) performed with the source substances Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0) and 2,3-dihydroxypropyl oleate (CAS 111-03-5) at dose levels up to and including 1000 mg/kg bw/day.

The potential of a substance to be absorbed from the gastrointestinal tract may be influenced by several parameters, like chemical changes taking place in gastrointestinal fluids, as a result of metabolism by gastrointestinal flora, by enzymes released into the gastrointestinal 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 in some cases no longer apply (ECHA, 2014).

In general, mono-, di- and triglycerides (e.g. from dietary fat) undergo hydrolysis by lipases (a class of ubiquitous carboxylesterases) prior to absorption (Lehninger et al., 1998). There is sufficient evidence to assume that mono-, di- and triglycerides in general will likewise undergo enzymatic hydrolysis in the gastrointestinal tract as the first step in their absorption, distribution, metabolism and excretion (ADME) pathways.

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., 1967; 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 substance Glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3), rapid ester hydrolysis in intestinal fluid simulant was confirmed (see IUCLID 6 Section 7.1.1).

Dub TGI 24 is therefore predicted to be enzymatically hydrolysed to primarily glycerol and 2-decyltetradecanoic acid, and to 2 -monoacylglycerol.

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 will be reassembled, primarily by re-esterification of absorbed 2-monoacylglycerols. The free glycerol is readily absorbed and little of it is re-esterified. The absorption of short-chain fatty acids can begin already in the stomach. This is because, in general, for intestinal absorption short-chain or unsaturated fatty acids are more readily absorbed than long-chain, saturated fatty acids. The absorption rate 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 (see IUCLID 6 Section 7.1.1). The results of the study show 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, the absorption degree was higher than 80%.

Overall, the target substance Dub TGI 24 is predicted 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 expected to be moderate-high.

In conclusion, the available information indicates that the hydrolysis products of Dub TGI 24 will have a moderate-high oral absorption.

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. 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, 2014).

Dub TGI 24 is a liquid, which favours dermal absorption. However, the low water solubility, high log Pow and molecular weight are in ranges that indicate a low to moderate absorption rate through the skin.

The dermal permeability coefficient (Kp) can be calculated from log Pow and molecular weight (MW) by applying the following equation described in US EPA (2004):

log(Kp) = -2.80 + 0.66 log Pow – 0.0056 MW

The Kp is calculated to be 22.7 cm/h for the constituent with the lowest MW, using the log Pow 10. Considering the water solubility (< 0.05 μg/cm³), the maximum dermal flux (calculated for the constituent with the lowest MW) is estimated to be approximately 1.135 μg/cm²/h and the dermal absorption potential is predicted as moderate.

No local or systemic effects were observed in the acute dermal toxicity study performed with the source substance glycerol tristearate (CAS 555-43-1) at a dose of 2000 mg/kg bw (please refer to IUCLID section 7.2.3). 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, 2014).

The available data provide no indications for skin irritating effects of Dub TGI 24 in rabbits. No skin effects were noted in the acute dermal toxicity study at the limit dose of 2000 mg/kg bw, performed with source substances glycerol tristearate (CAS 555-43-1) (please refer to IUCLID section 7.2.3). The result of the skin sensitisation tests (GMPT and RIPT in humans) performed with the source substances glycerol tristearate (CAS 555-43-1) and docosanoic acid ester with 1,2,3-propanetriol (CAS 77538-19-3), respectively (please refer to IUCLID section 7.4.1). Furthermore, no alerts for the mono-, di- and triglyceride of the target substance were predicted in the OECD QSAR Toolbox using the ‘Protein binding alerts for skin sensitisation’ in the OASIS v1.3 database (please refer to IUCLID section 7.4.1). Therefore, no enhanced penetration of the substance due to skin damage is expected. Taking all the available information into account, the dermal absorption potential is considered to be moderate.

Inhalation

Dub TGI 24 is a liquid with low vapour pressure (< 0.0001 Pa at 20 °C), and therefore low volatility. Under normal use and handling conditions, inhalation exposure and availability for respiratory absorption of the substance in the form of vapours, gases, or mists is considered to be limited (ECHA, 2014). However, the substance may be available for inhalatory absorption 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. Particles deposited in the nasopharyngeal/thoracic region will mainly be cleared from the airways by the mucocilliary mechanism and swallowed.

As no exposure to Dub TGI 24 via the inhalation route is predicted, the potential exposure is assumed to be negligible.

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, 2014).

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

Absorbed glycerol is readily distributed throughout the organism and it can be re-esterified to form endogenous triglycerides, be metabolised and incorporated into physiological pathways, like the glycolysis pathway (Lehninger, 1998). After being absorbed, fatty acids are (re-)esterified along with other fatty acids into triglycerides and released in chylomicrons into the lymphatic system. Fatty acids of carbon chain length ≤ 12 may be transported directly to the liver via the portal vein as the free acid bound to albumin, instead of being re-esterified. Chylomicrons are transported in the lymph to the thoracic duct and subsequently to the venous system. On 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 to derive energy or they are released into the systemic circulation and returned to the liver, where they are metabolised, stored or re-enter the circulation (IOM, 2005; Johnson, 1990; Johnson, 2001; Lehninger, 1998; NTP, 1994; Stryer, 1996; WHO, 2001).

There is a continuous turnover of stored fatty acids, as they are constantly metabolised to generate energy and then excreted as CO₂. Accumulation of fatty acids takes place only if their intake exceeds the caloric requirements of the organism.

In a study performed with 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester (surrogate of Glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3)), the systemic distribution of the radiolabelled material was assessed in rats (see IUCLID 6 Section 7.1.1). 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 the highest levels recovered in the gastrointestinal tract, liver and the remaining carcass. This shows that the substance was extensively absorbed from the gastrointestinal tract and distributed within the body. Due to excretion and absorption of the radiolabelled material, the radioactivity content in the gastrointestinal tract decreased rapidly from the 1-hr time point over the course of the study (168 hrs). This was similar for the radioactivity recovered in liver, which peaked at the 24-hr time point before decreasing gradually. The radioactivity found in the carcasses was nearly constant at the selected time points (app. 7%), indicating that the radiolabelled material may have been distributed to other tissues than the ones selected for analyses. The recovery of the radioactivity in excreta was >95% 72 hrs after administration, with the greatest amount of radioactivity eliminated via CO₂ (app. 77%). Based on the results of this study, no bioaccumulation potential was observed for 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester. This conclusion is considered to be applicable to the target substance, as well due to structural similarities and common functional groups.

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 most animal tissues and uses an enzyme complex for a series of oxidation- and hydration reactions, resulting in the cleavage of acetate groups in the form of acetyl-CoA. The alkyl chain length is reduced by 2 carbon atoms during each β-oxidation cycle. Alternative pathways for oxidation can be found in the liver (ω-oxidation) and the brain (α-oxidation). 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 will enter into the 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).

The potential metabolites following enzymatic metabolism of the test substance were predicted using the QSAR OECD toolbox (OECD, 2014). This QSAR tool predicts which primary and secondary metabolites of the test substance may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the gastrointestinal tract. Thirteen (13) to fifteen (15) hepatic metabolites and 10-25 dermal metabolites were predicted for the mono-, di- and triglyceride components. Primarily, the ester bond is broken both in the liver and in the skin, after which the hydrolysis products may be metabolised further. The resulting liver and skin metabolites are the products of alpha-, beta- or omega-oxidation (= addition of hydroxyl group). In the case of omega-oxidation, it is followed by further oxidation to the aldehyde, which is then oxidised to the corresponding carboxylic acid. For a branched fatty acid, the alpha- and omega pathways are particularly relevant. The ester bond may also remain intact, in which case a hydroxyl group is added to, or substituted with, a methyl group. In general, the hydroxyl groups make the substances more water-soluble and susceptible to metabolism by phase II-enzymes. The metabolites formed in the skin are expected to enter the blood circulation and have the same fate as the hepatic metabolites. Up to 183 metabolites were predicted to result from all kinds of microbiological metabolism. The high number includes many minor variations in the c-chain length and number of carbonyl- and hydroxyl groups; reflecting the diversity of microbial enzymes identified. Not all of these reactions are expected to take place in the human GI-tract. The results of the OECD toolbox simulation support the information on metabolism routes retrieved in the literature.

There is no indication that Dub TGI 24 is activated to reactive intermediates under the relevant test conditions. The experimental studies performed on genotoxicity (Ames test, gene mutation in mammalian cells in vitro, chromosome aberration assay in mammalian cells in vitro) using source substances were consistently negative, with and without metabolic activation. The result of the skin sensitisation studies performed in guinea pigs and humans using source substances were likewise negative.

 

Excretion

The non-absorbed fraction of Dub TGI 24 that is not hydrolysed in the gastrointestinal tract will be excreted via the faeces.

In general, the hydrolysis products glycerol and fatty acids are catabolised entirely by oxidative physiologic pathways, ultimately leading to the formation of carbon dioxide and water. Non-metabolised glycerol is a polar molecule and can readily be excreted via the urine. Small amounts of ketone bodies resulting from the oxidation of fatty acids may be excreted via the urine, however, the major part of the fatty acids will enter an oxidative pathway as described above under ‘Metabolism’ (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 hour period post-dose was very high (urine, 6.5%; faeces, 24.5%; CO, 77%; and cage wash, 0.5%). Most of the recovered radioactivity (97.5%, of which 71% CO, 21% faeces, 5.5% urine) was excreted up to and including the 24 hrs post-dose sampling time point (please refer to IUCLID section 7.1.1). The results confirm that glycerides, including Dub TGI 24, are mainly excreted as CO₂ in the expired air as a result of metabolism.

A detailed reference list is provided in the technical dossier (see IUCLID 6, section 13) and within the CSR.

References

Aungst B. and Shen D.D. (1986). Gastrointestinal absorption of toxic agents. In Rozman K.K. and Hanninen O. Gastrointestinal Toxicology. Elsevier, New York, US.

Barry, R.J.C. et al. (1967). Handling of glycerides of acetic acid by small rat intestine in vitro. J. Physiol., 185, 667-683

Cohen, M. et al. (1971). Lipolytic activity of human gastric and duodenal juice against medium and long chain triglycerides. Gastroenterology 60(1):1-15.

Cosmetic Ingredient Review Expert Panel (CIR) (1983). Final report on the safety assessment of Isostearic acid.J. of the Am. Coll. of Toxicol.2(7):61-74

Cosmetic Ingredient Review Expert Panel (CIR) (1987) Final report on the safety assessment of oleic acid, lauric acid, palmitic acid, myristic acid, stearic acid.J. of the Am. Coll. of Toxicol.6(3):321-401.

ECHA (2014). Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance. Version 2.0, November 2014. European Chemicals Agency, Finland.

Greenberger, N.J. et al. (1966). Absorption of medium and long chain triglycerides: factors influencing their hydrolysis and transport. J Clin Invest 45(2):217-27.

Institute of the National Academies (IOM) (2005). Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (Macronutrients). The National Academies Press. http://www.nap.edu/openbook.php?record_id=10490&page=R1

Johnson, R.C. et al. (1990). Medium-chain-triglyceride lipid emulsion: metabolism and tissue distribution. Am J Clin Nutr 52(3):502-8.

Johnson W Jr; Cosmetic Ingredient Review Expert Panel. (2001). Final report on the safety assessment of trilaurin, triarachidin, tribehenin, tricaprin, tricaprylin, trierucin, triheptanoin, triheptylundecanoin, triisononanoin, triisopalmitin, triisostearin, trilinolein, trimyristin, trioctanoin, triolein, tripalmitin, tripalmitolein, triricinolein, tristearin, triundecanoin, glyceryl triacetyl hydroxystearate, glyceryl triacetyl ricinoleate, and glyceryl stearate diacetate. Int J Toxicol. 2001;20 Suppl 4:61-94.

Lehninger, A.L., Nelson, D.L. and Cox, M.M. (1998).Prinzipien der Biochemie. 2. Auflage. Heidelberg Berlin Oxford: Spektrum Akademischer Verlag.

Lippel, K. (1973). Activation of branched and other long-chain fatty acids by rat liver microsomes. Journal of Lipid Research 14:102-109.

Mattson, F.H. and Volpenhein, R.A. (1962). Rearrangement of glyceride fatty acids during digestion and absorption. J Biol Chem 237:53-5.

Mattson, F.H. and Volpenhein, R.A. (1964). The digestion and absorption of triglycerides. J Biol Chem 239:2772-7.

Mattson, F.H. and Volpenhein, R.A. (1966). Carboxylic ester hydrolases of rat pancreatic juice. J Lipid Res 7(4):536-43.

Mattson, F.H. and Volpenhein, R.A. (1968). Hydrolysis of primary and secondary esters of glycerol by pancreatic juice. J Lipid Res 9(1):79-84.

National Toxicology Program (NTP) (1994) Comparative toxicology studies of Corn Oil, Safflower Oil, and Tricaprylin (CAS Nos. 8001-30-7, 8001-23-8, and 538-23-8) in Male F344/N Rats as vehicles for gavage. http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr426.pdf (2011-12-19). Report No.: C62215. Owner company: U.S. Department of Health and Human Services, Public Health Services, National Institutes of Health.

Stryer, L. (1996). Biochemie. 4. Auflage. Heidelberg Berlin Oxford: Spektrum Akademischer Verlag.

US EPA (2004).  Risk Assessment Guidance for Superfund (RAGS), Volume I: Human Health Evaluation Manual (Part E, Supplemental Guidance for Dermal Risk Assessment) Interim. http://www.epa.gov/oswer/riskassessment/ragse/index.htm

WHO (1967). Toxicological Evaluation of Some Antimicrobials, Antioxidants, Emulsifiers, Stabilizers, Flour-Treatment Agents, Acids and Bases: Acetic Acid and Fatty Acid Esters of Glycerol. FAO Nutrition Meetings Report Series No. 40A, B, C.

WHO (1974). Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants, emulsifiers and thickening agents: Acetic Acid and Its Potassium and Sodium Salts. WHO Food Additives Series No. 5.

WHO (1975). Toxicological evaluation of some food colours, thickening agents, and certain other substances: Triacetin. WHO Food Additives Series No. 8.

WHO (2001). Safety Evaluation of Certain Food Additives and Contaminants: Aliphatic Acyclic Diols, Triols, and Related Substances. WHO Food Additives Series No. 48.