Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption of soybean oil was measured in a 15-d feeding study by analysis of unabsorbed fecal fat. The net fat absorption was calculated from dietary intakes and fecal excretion.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Housing: The animals were housed in individual stainless steel wire-mesh cages
- Diet: Ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 50±5%
- Photoperiod (h dark / h light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Normal diet without any other fat source

Duration and frequency of treatment / exposure:

15 d; daily ad libitum in food

Remarks:

Doses / Concentrations:
15%

No. of animals per sex per dose / concentration:

10 animals/group

Control animals:

no

Details on study design:

- Rationale for animal assignment: Random

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: Feces
- Time and frequency of sampling: For the final 10 d, the feces were collected for analysing the unabsorbed fat. The net fat absorption was calculated from dietary intakes and fecal excretion.

Type:

absorption

Results:

94±2%

Details on absorption:

High absorption (94±2%) was observed for soybean oil.

Metabolites identified:

not measured

Conclusions:

Under the test conditions, the constituent was highly absorbed (94%) after dietary administration in rats.

Executive summary:

The oral absorption of the constituent 'glycerides, C16 -18 and C18 -unsatd.' (as soybean oil) was measured in a 15 d feeding study by analysis of fecal unabsorbed fat. Diet containing 15% of the constituent was fed to 10 male Sprague-Dawley rats for 15 d. For the final 10 d, the feces were collected for unabsorbed fat analysis. The net fat absorption was calculated from dietary intakes and fecal excretion. High (94%) absorption was observed. Under the test conditions, the constituent was highly absorbed after dietary administration in rats (Nolen, 1981 (1)).

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption of soybean oil was measured in a 91-d oral toxicity study by analysis of unabsorbed fecal fat. During the 3rd and 11th wks of study, feces were collected for analysis of unabsorbed fecal fat. The net fat absorption was calculated from dietary intakes and fecal excretion.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Housing: The animals were housed in individual stainless steel wire-mesh cages
- Diet: Ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 50±5%
- Photoperiod (h dark / h light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Normal diet without any other fat source

Duration and frequency of treatment / exposure:

91 d; daily ad libitum in food

Remarks:

Doses / Concentrations:
19%

No. of animals per sex per dose / concentration:

20 animals/sex/group

Control animals:

no

Details on study design:

- Rationale for animal assignment: Animals were distributed into groups of 20 rats per sex so that the litter mates were distributed evenly among the groups and the mean body weights did not vary more than 0.5 g.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: Feces
- Time and frequency of sampling: During the 3rd and 11th wks of study, feces were collected from 10 animals/sex/group for analysis of unabsorbed fecal fat. The net fat absorption was calculated from dietary intakes and fecal excretion.

Type:

absorption

Results:

96.5%

Details on absorption:

High absorption was observed for soybean oil (95±1% - males; 98±1% - females)

Key result

Test no.:

#1

Toxicokinetic parameters:

other: % absorption = 96.5 (mean value)

Metabolites identified:

not measured

Conclusions:

Under the test conditions, the constituent was highly absorbed (96%) after dietary administration in rats.

Executive summary:

The absorption of 'glycerides, C16-18 and C18-unsatd.' (as soybean oil) was measured in a 91 d oral toxicity study by analysis of unabsorbed fecal fat. Diet containing 19% test substance (7.5% experimental fat plus 11.5% as source of norma dietary fat) was fed to 20 Sprague-Dawley rats/sex for 91 d. During 3rd and 11th weeks of study, feces were collected from 10 animals/sex/group for analysis of unabsorbed fat. The net fat absorption was calculated from dietary intakes and fecal excretion. High (96%) absorption was observed. Under the test conditions, the test substance was highly absorbed after dietary administration in rats (Nolen, 1981 (2)).

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption of fully hydrogenated soybean oil was measured in a 15-d feeding study in rats by analysis of fecal unabsorbed fat.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Weight at study initiation: 200 g
- Housing: The animals were housed in individual stainless steel wire-mesh cages
- Diet: Ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 50±5%
- Photoperiod (h dark / h light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Normal diet without any other fat source

Duration and frequency of treatment / exposure:

15 d; daily ad libitum in food

Remarks:

Doses / Concentrations:
15%

No. of animals per sex per dose / concentration:

10 animals/sex/group

Control animals:

other: yes, 15% soybean oil

Details on study design:

- Rationale for animal assignment: Random

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: Feces
- Time and frequency of sampling: For the final 10 d, the feces were collected for unabsorbed fat analysis.

Statistics:

All of the data were analyzed by the Analysis of Variance and partitioned by the Tukey minimum significant difference method.

Details on absorption:

Low absorption was observed for fully hydrogenated soybean oil (6±4%) compared to soybean oil (94±2%).

Key result

Test no.:

#1

Toxicokinetic parameters:

other: % absorption = 6±4%

Metabolites identified:

not measured

Conclusions:

Under the test conditions, the constituent was poorly absorbed after dietary administration in rats.

Executive summary:

The oral absorption of the constituent 'glycerides, C16 -18' (as fully hydrogenated soybean oil) was measured in a 15 d feeding study by analysis of fecal unabsorbed fat. Diet containing 15% of the constituent was fed to 10 Sprague-Dawley rats/sex for 15 d. Control group was fed with 15% soybean oil for same duration. Feces were collected for the final 10 d, and analysed for unabsorbed fat. Low absorption was observed for the test substance (6±4% ) compared to soybean oil (94±2%). Under the test conditions, the constituent was poorly absorbed after dietary administration in rats (Nolen 1981 (1)).

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption of fully hydrogenated soybean oil was measured in a 91-d oral toxicity study by analysis of unabsorbed fecal fat.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Housing: The animals were housed in individual stainless steel wire-mesh cages
- Diet: Ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 50±5%
- Photoperiod (h dark / h light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Normal diet containing 11.5% soybean oil as fat source

Duration and frequency of treatment / exposure:

91 d; daily ad libitum in food

Remarks:

Doses / Concentrations:
7.5%

No. of animals per sex per dose / concentration:

20 animals/sex/group

Control animals:

other: yes, 19% soybean oil

Details on study design:

- Rationale for animal assignment: Animals were distributed into groups of 20 rats per sex so that the litter mates were distributed evenly among the groups and the mean body weights did not vary more than 0.5 g.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: Feces
- Time and frequency of sampling: During the 3rd and 11th wks of study, feces were collected from 10 animals/sex/group for analysis of unabsorbed fecal fat.

Statistics:

All of the data were analyzed by the Analysis of Variance and partitioned by the Tukey minimum significant difference method.

Type:

absorption

Results:

17±8% (males)

Type:

absorption

Results:

7±7% (females)

Details on absorption:

Low absorption was observed for experimental fat i.e. fully hydrogenated soybean oil (males: 17±8% ; females: 7±7% ). The total fat (i.e. fully hydrogenated soybean oil + soybean oil) absorption was significantly lower in treatment group (males: 64±4%; females: 68±3%) with respect to control group (males: 95±1%; females: 98±1%).

Metabolites identified:

not measured

Conclusions:

Under the test conditions, the constituent was poorly absorbed after dietary administration in rats.

Executive summary:

The oral absorption of the constituent 'glycerides, C16 -18' (as fully hydrogenated soybean oil) was measured in a 91 d oral toxicity study by analysis of unabsorbed fecal fat. Diet containing 7.5% of the constituent (plus 11.5% soybean oil as normal fat source) was fed to 20 Sprague-Dawley rats/sex for 91 d. Control group was fed with 19% soybean oil for same duration. During the 3rd and 11th weeks of study, feces were collected from 10 animals/sex/group for analysis of unabsorbed fat. Low absorption (17%) was observed for the substance in comparison to soybean oil. Under the test conditions, the constituent was poorly absorbed after dietary administration in rats (Nolen, 1981 (2)).

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The absorption of coconut oil was determined in a 47 wk repeated dose study in rats. At intervals during the study, feces were collected daily, pooled in weekly samples, and analyzed for fat content. The net fat absorption was calculated from dietary intakes and fecal excretion.

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Individual screen-bottom cages
- Diet: Normal diet; ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
Air-conditioned animal rooms

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (type of food): 18.5% coconut oil was mixed with diet containing additional 2.5% safflower oil as fat source to ensure adequacy of the essential fatty acids. No other fat source was used in diet.

Duration and frequency of treatment / exposure:

47 wks; ad libitum in diet

Remarks:

Doses / Concentrations:
18.5%

No. of animals per sex per dose / concentration:

15 animals/sex/group

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption)
- Tissues and body fluids sampled: Feces
- Details: At intervals during the study, feces were collected daily, pooled in weekly samples, and analyzed for fat content. The net fat absorption was calculated from dietary intakes and fecal excretion.

Type:

absorption

Results:

96% (high absorption)

Details on absorption:

The net fat absorption, calculated from dietary intakes and fecal excretion, was 96%. The absorption of coconut oil can be considered to be similar to this value as 2.5% safflower oil was the only other fat source in diet.

Metabolites identified:

not measured

Conclusions:

Under the test conditions, the constituent was highly absorbed after dietary administration in rats.

Executive summary:

The absorption of the constituent 'glycerides, C8-18 and C18-unsatd.' (as coconut oil) was determined in a 47 week repeated dose study in rats. The substance was included at 18.5% level in diets of 15 wistar rats/sex/group for 47 weeks. Additional 2.5% safflower oil was included to ensure adequacy of the essential fatty acids. At intervals during the study, feces were collected daily, pooled in weekly samples, and analyzed for fat content. The net fat absorption was calculated from dietary intakes and fecal excretion. The net fat absorption was 96%. The absorption of the substance can be considered to be similar to this value as 2.5% safflower oil was the only other fat source in diet. Hence, under the test conditions, the constituent was highly absorbed after dietary administration in rats (Harkins, 1968).

Reference 6

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The general plan of the experiment was to feed two groups of rats on a complete artificial diet, while one group received in addition a small quantity of squalene each day.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Diet: complete artificial diet that described by Drummond and Coward [1920].

Route of administration:

other: dropped directly in mouths from a micro-burette

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

Single daily administration for 42 days

Remarks:

660 mg

No. of animals per sex per dose / concentration:

Test group: 10 rats
Negative control: 9 rats

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection rationale: based on animal's weight

Type:

absorption

Results:

38.6%

Details on absorption:

About 10 g (approximately 38.6%) had been absorbed during the course of 42 days and this absorption had resulted in a marked increase in the weight of unsaponifiable matter in the livers and also in the bodies of these animals, namely, from 0.3680 to 1.0774 g in the case of the livers and from 1.8620 to 2.9114 g in that of the bodies. There was also a large increase in the amounts of cholesterol, from 0.2576 to 0.6189 g in the livers and from 1.3000 to 2.1620 g in the bodies.

Key result

Test no.:

#1

Transfer type:

secretion via gastric mucosa

Observation:

slight transfer

Details on excretion:

It was clear from the weight of unsaponifiable matter from the faeces of the latter animals, and from its iodine value, that much of the squalene administered had been excreted.

Metabolites identified:

not measured

It was assumed that that fraction of unsaponifiable matter which was not squalene had an iodine value which was the same as that of the material from the control group, namely 80.4%, then it may be calculated from the iodine value of the whole of this unsaponifiable matter (316), that the amount of squalene present was 81.1 %, i.e. 16.79 g (squalene has iodine value 371). This figure could only be approximate, but some rough check on it was obtained from the fact that, if the weight of material not sterol in the faeces of the control group substracted from the corresponding figure for the faeces of the animals receiving squalene, it may arrive at the figure 17.60 g. These figures 16.79 and 17.60 g can only be approximate, but they would seem to show that about 17 g of the 27.7 g of squalene administered had been excreted. Thus about 10 g (approximately 38.6%) had been absorbed during the course of 42 days and this absorption had resulted in a marked increase in the weight of unsaponifiable matter in the livers and also in the bodies of these animals, namely, from 0.3680 to 1.0774 g in the case of the livers and from 1.8620 to 2.9114 g in that of the bodies. There was also a large increase in the amounts of cholesterol, from 0.2576 to 0.6189 g in the livers and from 1.3000 to 2.1620 g in the bodies.

Conclusions:

Under the study conditions, the oral absorption of squalene was determined to be approximately 38.6% in rats.

Executive summary:

A study was conducted to determine effect of co-administration of squalene in subchronic repeated dose study by oral administration. The general plan of the experiment was to feed two groups of rats on a complete artificial diet, while one group received in addition a small quantity of squalene each day. Immediately before feeding-time the animals were given approximately 660 mg of squalene dropped directly into their mouths from a micro-burette. Records were taken of the amount of squalene given each day. The faeces were collected throughout the experiment at weekly intervals and stored in alcohol. At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible. No obvious ill effects followed the administration of squalene and post mortem examination showed that the organs of the animals were very healthy. There appeared to be a greater deposition of fat in the animals which received squalene and on the whole they seemed to grow at a somewhat greater rate than those of the control groups, possibly because the laxative action of the squalene caused a greater food consumption. It was assumed that approximately 17 g of the 27.7 g of squalene administered had been excreted and about 10 g (approximately 38.6%) have been absorbed during the course of 42 days. The experiment shows that when squalene is administered to the rat, it is in part absorbed and as a result of absorption there is a marked increase in the amounts of unsaponifiable matter and of cholesterol in the body and liver of the animal. Under the study conditions, the oral absorption of squalene was determined to be approximately 38.6% in rats (Channon, 1926).

Reference 7

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

metabolism

Principles of method if other than guideline:

A toxicokinetic study was conducted in rabbits to monitor the metabolism and distribution of squalene on exposure through sub-cutaneous route.

Species:

rabbit

Strain:

not specified

Sex:

not specified

Route of administration:

subcutaneous

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Twice daily for up to 12 d

Control animals:

not specified

Type:

metabolism

Results:

squalene is meatbolised to succinic and laevulinic acids along with small amounts of benzoic and hippuric acids

Type:

distribution

Results:

maximum storage was found in liver, muscle and skin

Details on distribution in tissues:

Considerable amount of squalene was found to be stored in liver, muscle, and skins of animals sacrificed either 4 h or 90 d after the last injection.

Metabolites identified:

yes

Details on metabolites:

Squalene was oxidized to succinic and laevulinic acids, in the body of rabbits. Urine samples also revealed presence of succinic acid, along with small amounts of benzoic and hippuric acids.

Conclusions:

Under the test conditions, the substance was found to be distributed and stored predominantly in liver, muscle and skin. It was metabolised to succinic, laevulinic acids and small amounts of benzoic and hippuric acids.

Executive summary:

Two different toxicokinetic studies were conducted to monitor the metabolism and distribution of the constituent squalene after exposure via the subcutaneous route in rabbits. In the study conducted to evaluate meatabolism, rabbits were injected subcutaneously (dose not specified) twice daily for up to 12 d. The substance was found to be oxidized to succinic and laevulinic acids. Urine samples also revealed the presence of succinic acid, along with small amounts of benzoic and hippuric acids. In the study, conducted to evaluate distribution, rabbits were sacrificed either 4 h or 90 d after the last injection. A considerable amount of the substance was found to be stored in liver, muscle and skin. Under the test conditions, the substance was found to be distributed and stored predominantly in liver, muscle and skin. It was metabolised to succinic, laevulinic acids and small amounts of benzoic and hippuric acids (CIR, 1982).

Reference 8

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

The dose-dependent absorption of the constituent (3-3H)stigmasterol was determined in a 24 h single dose study in rat. Absorption was evaluated by estimating 3H content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the constituent. Excretion of the constituent was determined by measuring 3H content of urine and faeces.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Charles River CD rat

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Fasting period before study: Yes, 24 h prior to and 2 h after dose administration
- Housing: In climate-controlled facility
- Individual metabolism cages: Yes, to permit the collection of urine, faeces and expired H2O
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 1 d

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil with added plant sterols containing unlabelled stigmasterol

Duration and frequency of treatment / exposure:

Frequency of treatment: single

Remarks:

Doses / Concentrations:
4, 40, 400 or 4000 mg/kg bw

No. of animals per sex per dose / concentration:

5 females/dose level

Control animals:

not specified

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, stomach and intestine contents, selected organs and whole body.
- Time and frequency of sampling: Samples were collected and analyzed for 3H at following intervals:
Urine: At 8 and 24 h
Faeces: At 24 h
Expired H2O: At 2, 4, 8 and 24 h
Analysis by whole body autoradiography: Single rat at 24 h after dosing

At 24 h, remaining animals were sacrificed and samples of blood, stomach and intestine contents, selected organs and the remaining carcasses were analysed for 3H.

Type:

absorption

Results:

Linear with respect to the dose of plant sterols and was of the order of 0.25% (based on regression line of absorbed stigmasterol upon administered total plant sterols)

Type:

distribution

Results:

Found in all tissues, although at very low levels with highest concentration at 24 h after dosing.

Type:

excretion

Results:

Mainly in faeces within 24 h, with trace amounts excreted in urine and exhaled air.

Details on absorption:

Absorption of (3-3H)stigmasterol after oral administration was linear with respect to the dose of plant sterols and was of the order of 0.25% (based on regression line of absorbed stigmasterol upon administered total plant sterols). Absorption was between 0.7 and 10% in tissues, with the highest level in the carcass at 24 h.

Details on distribution in tissues:

Autoradiography and tissue sample analysis revealed (3-3H)stigmasterol in all tissues, although at very low levels with highest in the carcass at 24 h after dosing. The tissue concentrations (expressed relative to wet weight) increased with dose. Distribution was similar in all dose levels, with highest radioactivity in the intestine, caecum and rectum. The adrenals and epithelia of the stomach and intestine contained highest tissue concentrations of 3H. Radioactivity was also present in the liver, bone marrow and ovary.

Key result

Transfer type:

other: Not determined

Details on excretion:

(3-3H)stigmasterol was excreted mainly in faeces within 24 h, with trace amounts excreted in urine and exhaled air.

Metabolites identified:

no

- Clinical Signs: No animals displayed any adverse clinical signs.

- HPLC and TLC analysis of faecal extracts: The constituent was excreted in both free and esterified forms.

Conclusions:

Under the test conditions, the consituent administered orally was only poorly absorbed; if absorbed, it was distributed into almost all tissues. It was excreted mainly via the faeces and to some extent via urine and expired air.

Executive summary:

A study was conducted to evaluate the absorption, distribution and excretion of the constituent (3 -3H)stigmasterol in Charles River CD rats. Four groups of 5 female rats each were treated with a single oral dose of the constituent (in sunflower oil with added plant sterols containing unlabelled test substance) at approximately 4, 40, 400 or 4,000 mg total sterol/kg bw. Absorption was evaluated by estimating 3H content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion of the substance was determined by measuring 3H content of urine and faeces. Absorption of the substance was linear with respect to the dose of plant sterols and was of the order of 0.25% (based on regression line of absorbed stigmasterol upon administered total plant sterols). Autoradiography and tissue sample analysis revealed that the constituent was found in all tissues, although at very low levels, with the highest 3H in the carcass at 24 h after dosing. The constituent was excreted mainly in faeces within 24 h, with trace amounts excreted in urine and exhaled air. Therefore, under the test conditions, the constituent administered orally was only poorly absorbed; if absorbed, it was distributed into almost all tissues. It was excreted mainly via the faeces and to some extent via urine and expired air (ANZFA, 2001 (1)).

Reference 9

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

The absorption, distribution and excretion of the constituent (3-3H)stigmasterol was determined in a single dose 96 h study in rats. Absorption was evaluated by estimating 3H content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the constituent. Excretion of the constituent was determined by measuring 3H content of urine and faeces.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Charles River CD rat

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Fasting period before study: Yes, 24 h prior to and 2 h after dose administration
- Housing: In climate-controlled facility
- Individual metabolism cages: Yes, to permit the collection of urine, faeces and expired H2O.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 1 day

Route of administration:

oral: gavage

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Frequency of treatment: Single dose

Remarks:

Doses / Concentrations:
1.9 mg/kg bw

No. of animals per sex per dose / concentration:

10 females/dose level

Control animals:

not specified

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, stomach and intestine contents, selected organs and whole body.
- Time and frequency of sampling: Samples were collected and analyzed for 3H at following intervals:
Urine : At 8, 24, 48, 72 and 96 h.
Faeces : At 24, 48, 72 and 96 h.
Expired H2O: At 2, 4, 8 and 24 h
Analysis by whole body autoradiography: Single rat at 8, 24, 48, 72 and 96 h after dosing
At 24 and 96 h, 3 and 2 rats respectively were sacrificed and samples of blood, stomach and intestine contents, selected organs and the remaining carcasses were analysed for 3H.

Type:

absorption

Results:

low (approximately 4.0 and 4.4% retained at 24 and 96 h respectively).

Type:

distribution

Results:

low levels in all tissues with maximum concentration in liver (0.4% at 24 h). There was a decline in tissue concentrations from 24 to 96 h.

Type:

excretion

Results:

85% and 87% was excreted in faeces within 24 and 96 h respectively; less than 1% was excreted in urine at 96 h and less than 0.1% of dose was expired in form of H2O.

Details on absorption:

Absorption of (3-3H)stigmasterol was low. Only about 4.0 and 4.4% of the absorbed substance was retained at 24 and 96 h respectively.

Details on distribution in tissues:

- Autoradiography and tissue sample analysis showed that the constituent was found at low levels in all tissues like adrenals, lungs, ovaries, stomach, uterus, brain, small and large intestine, spleen, heart, kidney and bone marrow.
- Maximum concentration was found in liver i.e., 0.4% at 24 h. There was a decline in tissue concentrations from 24 to 96 h.
- The gastrointestinal tract samples combined (excluding the caecum/rectum) contained less than 0.6% of dose at 24 h, with caecum and rectum containing 1.3%. At 96 h trace amounts of 3H were found in the intestinal tract.
- Concentrations of 3H were highest in the adrenals and the liver (expressed relative to wet weight at 24 h).

Key result

Transfer type:

other: Not determined

Details on excretion:

- 85% and 87% of test substance was excreted in faeces within 24 and 96 h respectively.
- A small proportion (less than 1%) was excreted in urine at 96 h.
- Less than 0.1% of dose was expired in form of H2O.

Metabolites identified:

no

- Clinical Signs: No animals displayed clinical signs of any adverse effects.

- HPLC and TLC analysis of faecal extracts: Test substance was excreted in both free and esterified forms.

Conclusions:

Under the test conditions, the constituent administered orally was only poorly absorbed. If absorbed, it was distributed into almost all tissues. It was excreted mainly via the faeces and to some extent via urine and expired air. 

Executive summary:

A study was conducted to evaluate the absorption, distribution and excretion of the constituent (3 -3H)stigmasterol in Charles River CD rat. Ten female rats received a single dose of the test substance at 1.9 mg/kg bw. Absorption was evaluated by estimating 3H content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the constituent. Excretion of the substance was determined by measuring 3H content of urine and faeces. Absorption of the substance was low (approximately 4.0 and 4.4% retained at 24 and 96 h respectively). Autoradiography showed that small amounts appeared in the adrenals, lungs, ovaries, stomach, uterus, brain, small and large intestine, spleen, heart, kidney and bone marrow. Approximately 87% of the administered dose was excreted in faeces within 96 h. A small proportion (less than 1%) was excreted in urine. Less than 0.1% of dose was expired in the form of H2O.  Hence, it can be concluded that the constituent administered orally was only poorly absorbed. If absorbed, it was distributed into almost all tissues. It was excreted mainly via the faeces and to some extent via urine and expired air (ANZFA, 2001 (2)).

Reference 10

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

The absorption, distribution and excretion of the constituent (3H)campesterol was determined in a 96 h single dose study in rats. Absorption was evaluated by estimating 3H content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the test material. Excretion of the substance was determined by measuring 3H content of urine and faeces.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Charles River CD rat

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Fasting period before study: Yes, 24 h prior to and for approximately 2 h after dose administration
- Housing: In climate-controlled facility
- Individual metabolism cages: Yes, to permit the collection of urine and faeces.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 1 d

Route of administration:

oral: gavage

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

1.7 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

10 females

Control animals:

not specified

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, stomach and intestine contents, selected organs and whole body.
- Time and frequency of sampling: Samples were collected and analyzed for 3H at following intervals:
Urine: At 8, 24, 48, 72 and 96 h
Faeces: At 24, 48, 72 and 96 h
Analysis by whole body autoradiography: Single rat at 8, 24, 48, 72 and 96 h after dosing
At 24 h and 96 h, 3 and 2 animals respectively were sacrificed and samples of blood, stomach and intestine contents, selected organs and the remaining carcasses were analysed for 3H.

Type:

absorption

Results:

low with approx. 13% retained at 24 h and approx. 10% after 96 h.

Type:

distribution

Results:

at 24 h, ditributed to all tissues; mainly in adrenals, spleen, intestinal epithelia, ovary, liver and bone marrow. At 96 h these tissue levels declined.

Type:

excretion

Results:

approx. 75 and 83% of the substance was excreted through faeces (major route) within 24 and 96 h respectively.

Details on absorption:

Absorption of (3H)campesterol was low with approx. 13 and 10% retained at 24 h and 96 h respectively..

Details on distribution in tissues:

Autoradiography showed that the substance was found in all tissues at 24 h (mainly in adrenals, spleen, intestinal epithelia, ovary, liver and bone marrow. ), and at 96 h all tissue levels declined.

Key result

Transfer type:

other: Not determined

Details on excretion:

The main route of excretion was through faeces with approx. 75 and 83% excretion within 24 and 96 h respectively. A small proportion (less than 0.2%) was excreted in urine in 96 h.

Metabolites identified:

no

- Clinical Signs: No animals displayed clinical signs of any adverse effects.

- HPLC and TLC analysis of faecal extracts: The constituent was excreted in both free and esterified forms.

Conclusions:

Under the test conditions, the constituent administered orally, was only poorly absorbed; The absorbed amount was distributed into all the tissues. It was excreted mainly via the faeces and to some extent via urine. 

Executive summary:

A study was conducted to evaluate the absorption, distribution and excretion of the constituent (3H) campesterol in Charles River CD rat. The substance was dosed to 10 female rats at 1.7 mg/kg bw by oral gavage. Absorption was evaluated by estimating 3H content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion was determined by measuring 3H content of urine and faeces. Absorption of the substance was low, with approximately 13% retained at 24 h and 10% after 96 h. Autoradiography showed that the substance was found in all tissues (mostly adrenals, spleen, intestinal epithelia, ovary, liver and bone marrow) at 24 h, but at 96 h all tissue levels declined. The main route of excretion was through faeces with approximately 75 and 83% excretion within 24 and 96 h, respectively.  A small proportion (less than 0.2%) was excreted in urine in 96 h. Under the test conditions, the substance administered orally, was only poorly absorbed; the absorbed amount was distributed into all the tissues. It was excreted mainly via the faeces and to some extent via urine (ANZFA, 2001 (1)).

Reference 11

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

metabolism

Principles of method if other than guideline:

The absorption, distribution, metabolism and excretion of the constituent (14C)β-sitosterol in rats was determined in a 96 h single dose study conducted in two separate experiments using different vehicles (sunflower and coconut oil). Absorption was evaluated by estimating 14C content in blood, stomach, intestine and several other organs. Whole body autoradiography was done to evaluate distribution of the test substance. Excretion of the substance was determined by measuring 14C content of urine and faeces.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Charles River CD rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Weight at study initiation: 152-193 g
- Fasting period before study: Yes, 24 h prior to and for approximately 2 h after dose administration
- Housing: In climate-controlled facility
- Individual metabolism cages: Yes, to permit the collection of urine and faeces.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 4 d

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil (experiment 1) & coconut oil (experiment 2)

Duration and frequency of treatment / exposure:

Single dose

Remarks:

Experiment 1: 3.0 mg/kg bw
Experiment 2: 55.0 mg/kg bw

No. of animals per sex per dose / concentration:

Experiment 1: 6/sex/dose level
Experiment 2: 2/sex/dose level; 1/sex/dose for coconut oil (vehicle control)

Control animals:

other: Yes, concurrent vehicle only in experiment 2 (coconut oil)

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, metabolism, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, stomach and intestine contents, selected organs and whole body
- Time and frequency of sampling: Samples were collected and analyzed for 14C at following intervals:
Experiment 1:
Urine: At sacrifice and at 8, 24, 48, 72 and 96 h
Faeces: At 24, 48, 72 and 96 h
Analysis by whole body autoradiography: Single rat from each sex and treatment group was sacrificed at 24, 48, and 96 h after dosing

Experiment 2:
Urine & faeces: At 8 and 24h
Total C content: At 24 h animals were sacrificed
At 96 h, remaining animals were sacrificed and samples of blood, stomach and intestine contents, selected organs and the remaining carcasses we re analysed for 14C.

Type:

absorption

Results:

approx. 5% of dose administered

Type:

distribution

Results:

at 24 h mainly seen in the intestinal tract, adrenal gland, ovary and less amount in the bone marrow, liver, intestinal lining and spleen. At 96 h, levels declined to background levels in almost all tissues

Type:

metabolism

Results:

a minor metabolite (not identified) was determined in the faeces in experiment 1

Type:

excretion

Results:

89% via faeces within 96 h and a small proportion in urine by 96 h.

Details on absorption:

Based on the urinary excretion data, and the small amounts of radioactivity appearing in tissues, it was considered that the absorption was approx. 5% of dose administered.

Details on distribution in tissues:

Autoradiography at 24 h showed that levels of 14C were associated with the intestinal tract, with smaller amounts appearing in the adrenal gland and ovary, and less in the bone marrow, liver, intestinal lining and spleen. At 96 h, 14C had declined to near background levels in all tissues except the adrenal gland and ovary.

Key result

Transfer type:

other: Not determined

Details on excretion:

In experiment 1, excretion of (14C)β-sitosterol was predominantly (over 89%) via faeces within 96 h, with over 70% and 80% of the administered dose excreted in the first 24 h in females and males respectively. A small proportion (0.2% and 0.1% in females and males respectively) was excreted in urine by 96 h.
Experiment 2: The predominant sterols in faeces were β-sitosterol, campesterol and cholesterol.

Metabolites identified:

no

Details on metabolites:

In experiment 1, a minor metabolite was determined in the faeces. However, its identity was not determined.

Clinical Signs: No animals displayed clinical signs of any adverse effects.

HPLC and TLC analysis of faecal extracts:

The substance was excreted in both free and esterified forms but the degree of esterification of free sitosterol was greater in Experiment 2.

This suggested that trans-esterification of sterols occurred in the gut. The predominant sterols in faeces were β-sitosterol, campesterol and cholesterol.

Conclusions:

Under the test conditions, the constituent administered orally to rat was only poorly absorbed. The absorbed amount was distributed into intestinal tract, adrenal gland, ovary, bone marrow, liver, spleen and adrenals. Extent of metabolism was small and the substance was excreted mainly via the faeces and urine.

Executive summary:

A study was conducted in two separate experiments using different vehicles (sunflower and coconut oil) to evaluate the absorption, distribution, metabolism and excretion of the constituent (14C)β-sitosterol in Charles River CD rat. In Experiment 1, 6 male and 6 female rats were given a single dose of 3.0 mg/kg of the substance in sunflower oil. In Experiment 2, 2 male and 2 female rats were given a single dose of 55.0 mg/kg of the substance in coconut oil with 1 animal/sex as concurrent coconut oil control. Absorption was evaluated by estimating 14C content of blood, stomach, intestine and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion of the substance was determined by measuring 14C content of urine and faeces. In both experiments absorption was approx. 5% of the administered dose. In experiment 1, autoradiography at 24 h showed that levels of 14C was associated with the intestinal tract, with smaller amounts appearing in the adrenal gland and ovary, and less in the bone marrow, liver, intestinal lining and spleen. At 96 h, 14C had declined to near background levels in all tissues except the adrenal gland and ovary. Excretion of (14C)β-sitosterol was predominantly (over 89%) via faeces within 96 h. A small proportion (0.2% and less than 0.1% in females and males respectively) was excreted in urine by 96 h. The substance was excreted in both free and esterified forms in both the experiments. Under the test conditions, the substance administered orally was only pooly absorbed. If absorbed, it was distributed into intestinal tract, adrenal gland, ovary, bone marrow, liver, spleen and adrenals. The substance was metabolized to some extent and excreted mainly via the faeces and urine (ANZFA, 2001 (1)).

Reference 12

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

metabolism

Principles of method if other than guideline:

The absorption, distribution, metabolism and excretion of the constituent (14C)β-sitosterol was determined in a 96 h single dose study in rats. Absorption was evaluated by estimating 14C content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion of the substance was determined by measuring 14C content of urine, faeces and expired air.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Charles River CD rat

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Weight at study initiation: 146-172 g
- Fasting period before study: Yes, 24 h prior to and for approximately 2 h after dose administration
- Housing: In climate-controlled facility
- Individual metabolism cages: Yes, to permit the collection of urine, faeces and expired carbon dioxide.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 4 d

Route of administration:

oral: gavage

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

0.6 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

10 rats

Control animals:

not specified

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, metabolism, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, stomach and intestine contents, selected organs and whole body.
- Time and frequency of sampling: Samples were collected and analyzed for 14C content at following intervals:
Urine and faeces : At sacrifice and at 8, 24, 48, 72 and 96 h.
Expired air (CO2): At 2, 4, 8 and 24 h.

Analysis by whole body autoradiography: Single rat from each treatment group was sacrificed at 4, 8, 24, 48, 72 and 96 h after dosing
At 96 h, remaining animals were sacrificed and samples of blood, stomach and intestine contents, selected organs and the remaining carcasses were analysed for 14C.

Type:

absorption

Results:

low (approximately 1.5% in all tissues, carcass and urine at 96 h)

Type:

distribution

Results:

small amounts appeared in the intestinal tract, liver, adrenals and other tissues

Type:

metabolism

Results:

minor unidentified metabolite was detected in faecal samples

Type:

excretion

Results:

94% of the administerd cosntituent was excreted through faeces within 96 h and less than 1% through urine. No evidence of excretion was found in expired air.

Details on absorption:

Absorption of the substance was low (approximately 1.5% in all tissues, carcass and urine at 96 h). The recovered (14C) was absent or very low in all tissues evaluated.

Details on distribution in tissues:

Autoradiography showed that levels of 14C from the substance were generally associated with the intestinal tract, small amounts in the liver, adrenals and other tissues from 4 h, declining to background levels at 72 h. The adrenal gland retained small amounts of radioactivity at 96 h.

Key result

Transfer type:

other: Not determined

Details on excretion:

The substance was excreted 94% in faeces within 96 h. A small proportion (less than 1%) was excreted in urine during this time. There was little evidence for significant biliary excretion of absorbed substance from whole body autoradiography. No evidence of excretion was found in expired air as 14CO2.

Metabolites identified:

no

Details on metabolites:

There was insufficient evidence to suggest hepatic metabolites. Minor unidentified metabolite was also detected in faecal samples.

Clinical Signs: No animals displayed clinical signs of any adverse effects.

HPLC and TLC analysis of faecal extracts: The substance was excreted in both free and esterified forms. The identity of these products was not determined and they may have represented other trans-esterified products formed in the gut.

Conclusions:

It can be concluded that the substance administered orally was only absorbed to a small extent; the absorbed amount was distributed into intestinal tract, liver and adrenals. It was metabolized to some extent and excreted mainly via the faeces and to some extent via urine. 

Executive summary:

A study was conducted to evaluate the absorption, distribution and excretion of the constituent (14C)β-sitosterol in the Charles River CD rat. Ten male rats were administered a single oral gavage dose of the test substance at 0.6 mg/kg bw. Absorption was evaluated by estimating 14C content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion of the substance was determined by measuring 14C content of urine, faeces and expired air. Absorption of the substance was low (approximately 1.5% in all tissues, carcass and urine). Autoradiography showed that small amounts of the substance appeared in the intestinal tract, liver, adrenals and other tissues. Approximately 94% of the administered dose was excreted in faeces within 96 h and a small proportion (less than 1%) was excreted in urine. The substance was excreted in both free and esterified forms. A minor, unidentified metabolite was also detected in faecal samples. No evidence of excretion was found in expired air. Under the study conditions, the substance administered orally was only low absorbed; if absorbed it was distributed into intestinal tract, liver and adrenals. It was metabolized to some extent and excreted mainly via the faeces and to some extent via urine (ANZFA, 2001 (2)). 

Reference 13

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

The absorption, distribution and metabolism of the constituent (14C)β-sitosterol was determined in a 24 h single dose study in rats. Absorption was evaluated by estimating 14C-labelled substance content in blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion of the substance was determined by measuring 14C content of urine and faeces.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Charles River CD rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Fasting period before study: Yes, 24 h prior to and for approximately 2 h after dose administration
- Housing: In climate-controlled facility
- Individual metabolism cages: Yes, to permit the collection of urine and faeces.
- Diet : Ad libitum
- Water : Ad libitum
- Acclimation period: 1 d

Route of administration:

oral: gavage

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

7.2 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

5 rats per sex

Control animals:

not specified

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, stomach and intestine contents, selected organs and whole body.
- Time and frequency of sampling: Samples were collected and analyzed for 14C at following intervals:
Urine: At 8 and 24 h
Faeces: At 24 h
At 24 h, remaining animals were sacrificed and samples of blood, stomach and intestine contents, selected organs and the remaining carcasses were analysed for 14C.

Type:

absorption

Results:

4.3% and 1.9% in females and males respectively.

Type:

distribution

Results:

adrenal glands and stomach of both sexes, and the ovaries of females were the primary sites of accumulation.

Type:

excretion

Results:

at 24 h, approx. 85% and 96% in females and males respectively was excreted through faeces and a small proportion (males 0.02% and females 0.07%) in urine.

Details on absorption:

The extent of absorption were 4.3% and 1.9% in females and males respectively.

Details on distribution in tissues:

- The substance was found in all tissues except the brain and testes; higher concentration (expressed as relative to wet weight of tissues) was found in females than in males.
- The adrenal glands and stomach of both sexes, and the ovaries of females were the primary sites of accumulation.
- Brain, heart kidney, uterus and testes had tissue levels below that of blood.

Key result

Transfer type:

other: Not determined

Details on excretion:

By 24 h, excretion of the constituent (14C)β-sitosterol was mainly via faeces with approx. 85% and 96% excretion in females and males respectively. A small proportion of 0.02% in males and 0.07% in females was excreted in urine.

Metabolites identified:

no

Clinical Signs: No animals displayed clinical signs of any adverse effects.

HPLC and TLC analysis of faecal extracts: The substance was excreted in modified forms which may be other trans-esterified products formed in the gut. A third minor HPLC peak seen following the dosing was also not characterised but may represent a product of oxidation, either in vivo or during sample storage.

Conclusions:

Under the test conditions, the substance administered orally was only poorly absorbed. The absorbed substance was distributed into adrenal glands, stomach of both sexes and the ovaries of females. It was excreted mainly via the faeces and to some extent via urine. 

Executive summary:

A study was conducted to evaluate the absorption, distribution and excretion of the constituent (14C)β-sitosterol in Charles River CD rats. The absorption, distribution and metabolism of the substance was determined in a 24 h single dose study in rats. The substance was dosed to 5 rats/sex at 7.2 mg/kg bw by oral gavage. Absorption was evaluated by estimating 14C content of blood, stomach, intestine contents and several other organs. Whole body autoradiography was done to evaluate distribution of the substance. Excretion of the substance was determined by measuring 14C content of urine and faeces. The absorption was 4.3 and 1.9% in females and males respectively. The absorbed substance was found in all tissues except the brain and testes. The adrenal glands and stomach of both sexes, and the ovaries of females were the primary sites of accumulation. At 24 h, excretion of (14C)β-sitosterol was mainly via faeces with approx. 85 and 96% excretion in females and males respectively. A small proportion was excreted in urine (males 0.02% and females 0.07%). It can be concluded that the substance administered orally was only poorly absorbed. The absorbed substance was distributed into adrenal glands, stomach of both sexes and the ovaries of females. It was excreted mainly via the faeces and to some extent via urine (ANZFA, 2001 (3)).

Reference 14

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Principles of method if other than guideline:

The metabolism (digestibility) of plant sterols was determined by incubating plant sterol with porcine cholesterol esterase or pancreatic lipase enzyme preparations for 1 to 24 h.

GLP compliance:

yes

Radiolabelling:

yes

Species:

other: in vitro test system

Route of administration:

other: in vitro

Vehicle:

not specified

Remarks:

Not specified

Control animals:

no

Type:

metabolism

Results:

Porcine cholesterol esterase and pancreatic lipase enzyme preparations were able to hydrolyse plant sterols.

Key result

Transfer type:

other:

Observation:

other: not applicable

Metabolites identified:

not measured

Details on metabolites:

Porcine cholesterol esterase and pancreatic lipase enzyme preparations were able to hydrolyse plant sterol. The rate of hydrolysis was greater with cholesterol esterase than with the lipase. This indicated that sterol esters will probably be hydrolysed in vivo in the intestinal tract.

Conclusions:

Under the test conditions, the substances were hydrolysed by porcine cholesterol esterase or pancreatic lipase and thus they are most likely to be hydrolysed in vivo in the intestinal tract.

Executive summary:

A study was conducted to evaluate the digestibility of plant sterols (mixture of stigmasterol, sitosterol, campesterol, 8.4% free sterols). Plant sterols were incubated with porcine cholesterol esterase or pancreatic lipase enzyme preparations for 1 to 24 h and were quantitated by HPLC, radio- HPLC and liquid scintillation after solvent extraction. Porcine cholesterol esterase and pancreatic lipase enzyme preparations were able to hydrolyse the substancee. The rate of hydrolysis was greater with cholesterol esterase than with the lipase. This indicated that sterol esters will probably be hydrolysed in vivo in the intestinal tract. Hence, it can be concluded that the substance are hydrolysed by porcine cholesterol esterase or pancreatic lipase and thus are most likely to be hydrolysed in vivo in the intestinal tract (ANZFA 2001 (2)).

Reference 15

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Objective of study:

excretion

metabolism

Principles of method if other than guideline:

The metabolism and excretion of alpha-tocopherol was evaluated in two separate studies in the JECFA report:
- Predominant route of excretion was evaluated in rats through exposure to 3.5 mg daily for one month.
- Metabolism aspect was evaluated in rats through exposure to single oral dose of 450 mg/kg bw

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

oral: capsule

Vehicle:

not specified

Duration and frequency of treatment / exposure:

Excretion study: Daily for one month
Metabolism study: Single oral dose

Remarks:

Doses / Concentrations:
Excretion study: 3.5 mg
Metabolism study: 450 mg/kg bw

Control animals:

not specified

Type:

excretion

Results:

3 to 15% of tocopherol appeared in the faeces at 3.5 mg of daily administration for one month

Type:

metabolism

Results:

Significant increase in aminopyrine demethylase activity in the liver was observed with alpha-tocopherol given as a single oral dose of 450 mg/kg

Details on excretion:

- 3 to 15% of tocopherol appeared in the faeces and there was no urinary excretion of tocopherol
- With larger doses, up to 25% appeared in the faeces

Metabolites identified:

not specified

Additional toxicokinetic studies from the JECFA evaluation report:

- Excretion: In another study conducted with labelled tocopherol, one or more metabolites are excreted in the urine (Sternberg & Pascoe-Dawson, 1959).

- Distribution: When more than the daily requirement is administered, there is some storage of tocopherol in the liver (Sebrell & Harris, 1972).

- Metabolism: alpha-tocopherol regulates certain microsomal hydroxylations (Carpenter, 1972).

Conclusions:

In the excretion study, maximum 15% of the constituent was excreted in the faeces, with no urinary excretion. In the metabolism study, a significant increase in aminopyrine demethylase activity in the liver was observed.

Executive summary:

The metabolism and excretion of the constituent alpha-tocopherol was evaluated in two separate studies:

Excretion study: 5 mg of the constituent was administered daily for one month to rats. Approximately 3 to 15% the substance was excreted in the faeces and none via urine. 

Metabolism study: A single oral dose of 450 mg/kg bw of the constituent was administered to rats. A significant increases in aminopyrine demethylase activity in the liver was observed.

Hence, it can be concluded that, in the excretion study, maximum 15% of the constituent was excreted in the faeces, with no urinary excretion. In the metabolism study significant increase in aminopyrine demethylase activity in the liver was observed with alpha-tocopherol (JECFA, 1987).

Reference 16

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not available

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

Principles of method if other than guideline:

A study was conducted to investigate the distribution of the constituent dl-alpha-tocopherol in groups of 10 fasted female albino rats, dosed by stomach tube with 4 mL of an emulsion containing ethanolic solutions of 2 mg dl-alpha-tocopheryl acetate and 50 µCi dl-alpha-tocopheryl-[1’,2’-3H2]-acetate. Two animals per group were killed 3, 6, 12, 24 or 48 h after dosing.

Radiolabelling:

yes

Species:

rat

Strain:

other: albino rats

Sex:

female

Route of administration:

oral: gavage

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 4 mL of an emulsion containing ethanolic solutions of 2 mg dl-alpha-tocopheryl acetate or 50 µCi dl-alpha-tocopheryl-[1’,2’-3H2]-acetate

VEHICLE: Ethanol

Duration and frequency of treatment / exposure:

48 h

Remarks:

Doses / Concentrations:
2 mg dl-alpha-tocopheryl acetate and 50 µCi dl-alpha-tocopheryl-[1’,2’-3H2]-acetate

No. of animals per sex per dose / concentration:

10 females

Control animals:

not specified

Details on dosing and sampling:

- Groups of 10 fasted female albino rats were dosed by stomach tube with 4 mL of an emulsion containing ethanolic solutions of 2 mg dl-alpha-tocopheryl acetate and 50 µCi dl-alpha-tocopheryl-[1’,2’-3H2]-acetate.
- Two animals per group were killed 3, 6, 12, 24, or 48 h after dosing

Type:

distribution

Results:

Liver was the principal storage site of tocopherol, but the adrenal glands had the greatest accumulation of radioactivity

Details on distribution in tissues:

- After 12 h, 47 to 86% of the radioactivity found in the blood, liver, spleen, kidneys, heart, ovaries, adipose tissue and skeletal muscle was recovered as tocopheryl quinone and 79% of the radioactivity found in the adrenal glands was as tocopherol.
- After 24 h, 82% of the radioactivity recovered in the adrenal glands and 66% recovered in skeletal muscle was as tocopherol; in each of the remaining examined tissues, 52% to 83% of the radioactivity was recovered as tocopheryl quinone.

Hence, the liver was the principal storage site of tocopherol, but the adrenal glands had the greatest accumulation of radioactivity (expressed as dpm/g wet tissue).

Metabolites identified:

not measured

Conclusions:

Under the test conditions, liver was the principal storage site of the constituent but the adrenal glands had the greatest accumulation of radioactivity.

Executive summary:

A study was conducted to investigate the distribution of the constituent alpha-tocopherol in albino rats after oral administration. Groups of 10 fasted female albino rats were dosed by stomach tube with 4 mL of an emulsion containing ethanolic solutions of 2 mg dl-alpha-tocopheryl acetate and 50 µCi dl-alpha-tocopheryl-[1’,2’-³H₂]-acetate. Two animals per group were killed 3, 6, 12, 24 or 48 h after dosing. After 12 h, 47 to 86% of the radioactivity found in the blood, liver, spleen, kidneys, heart, ovaries, adipose tissue and skeletal muscle was recovered as tocopheryl quinone and 79% of the radioactivity found in the adrenal glands was as tocopherol. After 24 h, 82% of the radioactivity recovered in the adrenal glands and 66% recovered in skeletal muscle was as tocopherol; in each of the remaining examined tissues, 52 to 83% of the radioactivity was recovered as tocopheryl quinone. Under the test conditions, liver was the principal storage site of the constituent but the adrenal glands had the greatest accumulation of radioactivity (Fiume, 2002 (2)).

Reference 17

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

Principles of method if other than guideline:

The oral absorption of the constituent tocopherol in terms of serum concentration was determined in 20 subjects following ingestion of two capsules (800 IU i.e. equivalent to 536 mg) of d-tocopherol preparation. Concentrations in blood samples were measured once prior to dosing and at 8, 24, and 48 h after dosing.

Radiolabelling:

no

Species:

human

Strain:

not specified

Sex:

male/female

Route of administration:

oral: capsule

Vehicle:

other: not applicable

Details on exposure:

Twenty subjects, 11 males and 9 females, were given two capsules (800 IU i.e. equivalent to 536 mg) of d-tocopherol preparation with 100 mL of whole milk; the milk was used to provide fat for the formation of chylomicrons.

Duration and frequency of treatment / exposure:

48 h

Remarks:

Doses / Concentrations:
536 mg

No. of animals per sex per dose / concentration:

Twenty subjects, 11 males and 9 females

Control animals:

not specified

Details on dosing and sampling:

- Twenty subjects, 11 males and 9 females, were given two capsules (800 IU i.e. equivalent to 536 mg) ) of d-tocopherol preparation with 100 mL of whole milk; the milk was used to provide fat for the formation of chylomicrons.
- Blood samples were taken prior to dosing and at 8, 24, and 48 h after dosing.

Type:

absorption

Results:

71% increase of alpha-tocopherol above baseline

Details on absorption:

- The peak mean serum concentration of d-tocopherol was observed after 8 h (1.71 mg/dL compared to baseline of 0.95 mg/dL).
- After 24 h, a statisically significant difference in the percentage increase of d-tocopherol above baseline (71%) was observed.

Metabolites identified:

not measured

Conclusions:

Under the test conditions, the peak mean serum concentration of the constituent was observed after 8 h (1.71 mg/dL compared to baseline of 0.95 mg/dL). After 24 h, a statisically significant difference in the percentage increase of the constituent above baseline (71%) was observed.

Executive summary:

The serum concentration of the constituent d- tocopherol was determined in 20 subjects after ingestion of two capsules (800 IU i.e. equivalent to 536 mg) of a d-tocopherol preparation and blood sample collection once prior to dosing and at 8, 24 and 48 h after dosing. The peak mean serum concentration of the constituent was observed after 8 h (1.71 mg/dL compared to baseline of 0.95 mg/dL). After 24 h, a statistically significant difference in the percentage increase of the constituent above baseline (71%) was observed (Fiume, 2002 (1)).

Reference 18

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

The penetration of the constituent alpha-tocopherol was determined using three human abdominal skin samples and three Skh:HR-1 mouse dorsal skin samples.

Radiolabelling:

no

Species:

other: in vitro

Duration of exposure:

24 h

Doses:

5% alpha-tocopherol (0.12 M)

No. of animals per group:

In vitro; human and mice skin sample

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human abdominal skin and three Skh:HR-1 mouse dorsal skin samples

PRINCIPLES OF ASSAY
- Diffusion cell: Franz-type diffusion cells

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

- The average cumulative penetration through human skin for tocopherol was 14 and 227 µg/cm2 after 2 and 24 h respectively.
- Using mouse skin, the average cumulative penetration of tocopherol was 50 and 628 µg/cm2 after 2 and 24 h, respectively.

Conclusions:

Under the study conditions, penetration of the constituent through mouse skin was approximately three times greater than that through human skin.

Executive summary:

A study was conducted to determine the dermal penetration potential of the constituent alpha-tocopherol using three human abdominal skin samples and three Skh:HR-1 mouse dorsal skin samples. The skin samples were mounted in Franz-type diffusion cells, with 0.785 cm² skin surface area exposed. The average cumulative penetration through human skin for the substance was 14 and 227 µg/cm2 after 2 and 24 h respectively. Using mouse skin, the average cumulative penetration of the test substance was 50 and 628 µg/cm2 after 2 and 24 h, respectively. Under the study conditions, penetration of the constituent through mouse skin was approximately three times greater than that through human skin (Fiume, 2002).

Reference 19

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A study was conducted in human volunteers to determine whether topically applied tocopheryl acetate was substantially absorbed into skin and whether it gets converted into free tocopherol. 

Radiolabelling:

not specified

Species:

human

Sex:

male/female

Type of coverage:

open

Vehicle:

not specified

Duration of exposure:

3 months

Doses:

125 mg/g in a vehicle cream

No. of animals per group:

Eleven humans (eight males and three females)

Details on study design:

The constituent was supplied in a vehicle cream at a concentration of 125 mg/g. The subjects applied the cream to their forearms twice daily for 3 months. Clinical evaluation, blood sampling, and 3-mm punch biopsies were performed prior to and at the end of treatment.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

- After 3 months, the mean concentrations of tocopheryl acetate, alpha-tocopherol (hydrolysis metabolite) and gamma-tocopherol (hydrolysis metabolite) found in skin were 256.3±195.5, 36.3±20.9 and 4.4±2.3 µg/g respectively against the respective baseline values as 5.9±1.8, 38.9±17.9 and 6.0±3.9 µg/g.
- The plasma concentrations of tocopheryl acetate and alpha-tocopherol were 2.5±1.3 and 13.3±6.1 µg/g against the respective baseline values as 2.1±0.9 and 12.7±6.3 µg/g.

Tocopheryl acetate was not detected in three of the four pooled skin samples prior to application; a concentration of 23.6 µg/g tissue determined in one pooled skin sample was assumed to be related to prior use of a sunscreen-containing tocopheryl acetate.

Conclusions:

Under the test conditions, the absorption of the constituent was substantial, but systemic availability was not observed. Also, systemic biotransformation to its unesterified form did not occur.

Executive summary:

A study was conducted in human volunteers to determine whether topically applied constituent tocopheryl acetate was substantially absorbed into skin and whether it converted into free tocopherol. Eleven subjects (eight males and three females), that had “at least three discrete clinically diagnosable keratoses” on their forearms were used in a study. The constituent was supplied in a vehicle cream at a concentration of 125 mg/g. The subjects applied the cream to their forearms twice daily for 3 months. Clinical evaluation, blood sampling, and 3 -mm punch biopsies were performed prior to and at the end of treatment. After 3 months, the mean concentrations of the tocopheryl acetate, alpha-tocopherol and gamma-tocopherol (the hydrolysis metabolites) found in skin, were 256.3±195.5, 36.3±20.9 and 4.4±2.3 µg/g respectively against the respective baseline values as 5.9±1.8, 38.9±17.9 and 6.0±3.9 µg/g. The plasma concentrations of tocopheryl acetate and alpha-tocopherol were 2.5±1.3 and 13.3±6.1 µg/g against the respective baseline values as 2.1±0.9 and 12.7±6.3 µg/g. Hence, absorption of the constituent was substantial, but systemic availability was not observed. Also, systemic biotransformation to its unesterified form (hydrolysis metabolites) did not occur (Fiume, 2002 (1)).

Reference 20

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Two studies were conducted to determine the amount of tocopherol and tocopheryl acetate in the epidermis after a single dose or repeated applications of 0.25% tocopheryl acetate.

Radiolabelling:

not specified

Species:

rat

Type of coverage:

open

Vehicle:

ethanol

Duration of exposure:

- For the single dose: 1 h
- For the daily applications: 5 h

Doses:

- For the single dose: 35 µL/cm2 of 0.25% tocopheryl acetate in ethanol was applied to shaved sites approximately 20 cm in diameter (i.e. approx. 1.673 mg, assuming 0.956 g/mL as the density of tocopheryl acetate)
- For the daily applications: 35 µL/cm2 of 0.25% tocopheryl acetate in ethanol was applied to shaved sites approximately 12 cm in diameter (i.e. approx. 1.0038 mg, assuming 0.956 g/mL as the density of tocopheryl acetate)

No. of animals per group:

- For the single dose: 5 rats
- For the daily applications: 10 rats

Control animals:

yes

Details on study design:

- For the single dose, 0.25% tocopheryl acetate in ethanol (35 µL/cm2) was applied to shaved sites approximately 20 cm in diameter on the backs of five rats. One animal was killed hourly after application, and skin samples were taken from the test site and an untreated site on the back.

- For the daily applications, 0.25% tocopheryl acetate in ethanol (35 µL/cm2) was applied to a shaved site approximately 12 cm in diameter on the left side of the back of 10 rats; ethanol was applied to the right side. Two animals were killed 5 hours after dosing, and skin samples were taken from the test and control sites. This procedure was repeated over the next 4 d.

Absorption in different matrices:

Single dose application and observation after 5 h:
- Five hours after pretreatment with ethanol (control), 17±4.9 ng/mg tocopherol was found in both the stratum corneum and viable layer. Concentrations of tocopherol and tocopheryl acetate after 5 h were 16±2 and 1920±330 ng/mg respectively from stratum corneum and 24±6, 540±300 ng/mg respectively from viable layers.

Repeated application and observation after 5 d:
After repeated applications for 5 d, concentrations of tocopherol and tocopheryl acetate were 42±4.3 and 4040±600 ng/mg respectively from stratum corneum and 102±19.7, 1510±160 ng/mg respectively from viable layers.

5 h after pretreatment with ethanol, 17±4.9 ng/mg tocopherol was found in both the stratum corneum and viable layer and hence the amount of endogenous tocopherol was 17±4.9 ng/mg.

Conclusions:

Under the test conditions, significant amount of absorption was revealed from the concentrations of the constituent in the viable layers after 5 h and repeated applications for 5 d. Further, measurable amount of tocopherol was not formed by hydrolysis after a single application of the constituent. With repeated applications of the substance, a small but steady increase was observed in the amount of tocopherol in the skin of treated and control sites.

Executive summary:

Two studies were conducted to determine the amount of the constituents tocopheryl acetate and its hydrolysis product in the epidermis after a single dose or repeated applications of 0.25% tocopheryl acetate. For the single dose, 0.25% tocopheryl acetate in ethanol (35µL/cm2) was applied to shaved sites approximately 20 cm in diameter on the backs of five rats (i.e. approx. 1.673 mg, assuming 0.956 g/mL as the density of tocopheryl acetate). One animal was killed hourly after application, and skin samples were taken from the test site and an untreated site on the back. For the daily applications, 0.25% tocopheryl acetate in ethanol (35µL/cm2) was applied to a shaved site approximately 12 cm in diameter on the left side of the back of 10 rats; ethanol was applied to the right side (i.e. approx. 1.0038 mg, assuming 0.956 g/mL as the density of tocopheryl acetate). Two animals were killed 5 h after dosing, and skin samples were taken from the test and control sites. This procedure was repeated over the next 4 d. 5 h after pretreatment with ethanol (control), 17±4.9 ng/mg tocopherol was found in both the stratum corneum and viable layer. Concentrations of tocopherol and tocopheryl acetate (test substance) after 5 h were 16±2 and 1920±330 ng/mg respectively from stratum corneum and 24±6, 540±300 ng/mg respectively from vaiable layers. After repeated applications for 5 d, concentrations of tocopherol and tocopheryl acetate (test substance) were 42±4.3 and 4040±600 ng/mg respectively from stratum corneum and 102±19.7, 1510±160 ng/mg respectively from viable layers. Under the test conditions significant amount of absorption was revealed from the concentrations of tocopherol acetate in the viable layers after 5 h and repeated applications for 5 d. Further, measurable amount of tocopherol was not formed by hydrolysis after a single application of the test substance. With repeated applications of the test substance, a small but steady increase was observed in the amount of tocopherol in the skin of treated and control sites (Fiume, 2002 (2)).

 

Reference 21

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

other: ADME

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

The absorption, distribution, and excretion of radiolabelled ethyl oleate (EO) was studied in Sprague-Dawley rats after a single, peroral dose of 1.7 or 3.4 g/kg body weight and was compared with a radiolabelled triacylglycerol (TG) containing only oleic acid as the fatty acid (triolein).

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

(14C) Ethyl oleate

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratory
- Age at study initiation: At dosing, the animals were approximately 8-10 weeks of age.
- Weight at study initiation: At dosing, the animals weighed 194-253 g.
- Housing: Housed individually in glass metabolism cages designed for the separation and collection of urine, feces, and expaired air.
- Individual metabolism cages: yes
- Diet :A certified standard rodent diet (Harlan Teklad) were available, ad libitum, at all times throughout the study period.
- Water :Water was available, ad libitum, at all times throughout the study period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature was controlled throughout the study.
- Humidity (%): Humidity was controlled throughout the study.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

Single dose

Remarks:

1.7 or 3.4 g/kg. Each animal received approximately 120 µCi/mg of radioactivity

No. of animals per sex per dose / concentration:

Twenty male and twenty female rats were divided into four groups, each group consisting of five rats per gender

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled :Adrenal glands, Bone (both femurs), Bone marrow (both), Brain, Carcass (residual), Cecum, Colon, Duodenum, Eyes (both), Fat (mesenteric), Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (mes), Muscles (thigh), Ovaries, Pancreas, Pituitary gland, Prostate, Rectum, Salivary glands, Spleen, Stomach, Testes, Thymus, Thyroid/parathyroids, Urine, feces and expired air.

- Time and frequency of sampling: Urine and feces were collected every 24 h until 72 h post-dose. Expired air was measured at 6, 12, 18, and 24 h post-dose. Animals were sacrificed at 72 h post-dose by exsanguination (cardiac puncture) under halothane anesthesia and tissues, including blood, were collected for anlaysis of radioactivity in ech matrix (See table 3 for details).

- Other: All samples were homogenised or mixed prior to radioanalysis, which was performed by either direct liquid scintillation counting (LSC) or by combustion followed by LSC, as appropriate. All samples were analysed in duplicate, if sample size allowed. Analysis was repeaed, sample size allowing, if the variability between the replicates was more than 10%.

Statistics:

Statistical analyses were limited to simple expressions of variation, such as mean and standard deviations

Type:

absorption

Results:

75-88%

Details on absorption:

Ethyl oleate was well absorbed with approximately 75-88 % of the dose absorbed.

Details on distribution in tissues:

Radioactivity (presumably in the form of oleic acid) recovered from all the tissues (including residual carcass) collected at sacrifice accounted for approximately 14-23% of the dose of (14C)EO. The males in all groups had a higher percentage of radioactivity in the tissues than did the corresponding females. In the treatment groups, the vast majority of radioactivity was found in the residual carcass (84-89% of the total tissue radioactivity). Aside from the residual carcass, the tissue with the highest percentage of radioactivity for all the groups was mesentric fat with levels ranging from 0.4-0.7% in the EO group. Tissue concentration were roughly dose-proportional with no remarkable-related differences. The overall mean blood to plasma concentration ratios for total radioactivity was very close to 1, thus indicating that radioactivity partitioned into both the cellular and plasma components of blood. With the exception of the brain and eyes, which had a tissue: plasma ratios of approximately 1 or less, the tissue to plasma ratio for all other tissues at sacrifice was higher than 1, indicating that (14C) EO- derived radioactivity had a tendency to accumulate in the tissue.

Details on excretion:

The cumulative excretion of radioactivity in each matrix per group is presented in table 2a and 2b for the lower (1.7 g/kg) and higher (3.4 g/kg) dose levels. The main route of excretion of radioactivity in the groups was via expired air as CO2. Excretion of (14C) CO2 was rapid in the groups such that by 12 h after dosing 40-70% of the administered dose was excreted in expired air. The females in the groups had a higher percentage of radioactivity expired as CO2 than did the corresponding males. A second route of elimination of radioactivity was via the feces in the groups. The mean percent dose recovered in the feces over the first 24 h post-dose was approximately 8 and 20% for the low and high doses of (14C) EO, respectively.
Renal elimination was minimal, with approximately 2% of the radioactivity recovered in urine over 72 h post-dose for the groups.

Metabolites identified:

yes

Details on metabolites:

Ethyl oleate (EO) is rapidly and extensively hydrolysed to free oleic acid, absorbed, and delivered to tissue where it undergoes β- oxidation. The basis for this conclusion is the rapid excretion of a significant percentage of the administered dose (30-40%) as CO2 within the first 6 h and 40-70% of the dose within 12 h. For this to happen, the free oleic acid moiety has to be available. At the 1.7 g/kg dose, the tissue distribution of EO-derived radioactivity was similar to that of triacylglycerol (TG)-derived radioactivity. Again, this supports the conclusion that EO is rapidly hydrolysed to oleic acid, absorbed, and distributed within the body in the same way as dietary sources of oleic acid. The similar tissue distribution between EO-derived radioactivity and TG-derived radioactivity suggests that the radiolabel in tissue represents the same chemical form (i.e., the oleic acid moiety).

It is likely that the vast majority of the actual chemical form of the ethyl oleate molecule absorbed is the oleic acid moiety. This is based on published in vivo studies which have demonstrated that EO and other fatty acid esters are rapidly hydrolysed to ethanol and free fatty acid within the GI tract(Froyland et al., 1996; Saghir et al.,199). Other supporting evidence for absorption of the free fatty acid (or very rapid in situ formation of the free acid) is the rapid excretion of radioactivity as CO2. This is because the radiolabel was located on the carboxyl carbon. Oxidation of fatty acids requires a free carboxyl end (i.e., not esterified). The rate and extent of 14CO2 extretion was similar between the EO and the TG groups indicating that both lipids deliver free fatty acid to tissue for oxidation at approximately the same rate and extent.

Table 2. The overall mean total recovery of radioactive dose by 72 h post-dose.

	Males (%)	Females (%)
Group 1 (Ethyl oleate 3.4 g/kg)	74.9	70.5
Group 2 (Ethyl oleate 1.7 g/kg)	87.5	87.3

 

Table 2A. Cumulative percent recovery of radioactivity following administration of a single oral dose of (¹⁴C) Ethyl oleate (1.7 g/kg) to males and females rats.

Collection intervals (h)	Percent of dose
	(^14C) Ethyl oleate (1.7 g/kg)
	Males		Females
	Mean (n=5) SD		Mean (n=5) SD
Urine
0-24	1.62	0.10	1.84	0.13
0-48	1.88	0.11	2.09	0.14
0-72	1.98	0.12	2.21	0.13
Feces
0-24	6.81	1.83	7.57	1.25
0-48	7.50	1.80	8.24	1.10
0-72	7.68	1.80	8.42	1.10
Expired air (carbon dioxide)
0-6	35.7	8.3	42.7	6.1
0-12	49.8	7.8	59.5	7.1
0-18	53.1	7.5	63.2	7.3
0-24	55.1	7.1	65.2	6.9
0-48	60.0	7.5	69.0	6.7
0-72	62.3	7.4	70.7	6.3
Tissues
72h	23.2	4.3	14.4	5.1
Mass balance^a	95.3	2.0	95.9	0.7

Table 2B. Cumulative percent recovery of radioactivity following administration of single dose of (¹⁴C) Ethyl oleate (3.4 g/kg) to male and female rats.

Collection intervals (h)	Percent of dose
	(^14C) Ethyl oleate (3.4 g/kg)
	Males		Females
	Mean (n=5) SD		Mean (n=5) SD
Urine
0-24	1.10	0.13	1.11	0.17
0-48	1.29	0.14	1.33	0.21
0-72	1.37	0.15	1.44	0.21
Feces
0-24	16.7	8.2	20.3	10.0
0-48	17.6	8.0	21.5	10.3
0-72	17.7	8.0	21.7	10.3
Expired air (carbon dioxide)
0-6	25.4	1.4	22.8	6.2
0-12	41.5	2.5	43.4	9.8
0-18	44.7	3.8	47.2	9.4
0-24	45.9	3.9	49.0	9.2
0-48	49.6	4.0	53.3	9.4
0-72	51.4	4.1	54.8	10.0
Tissues
72h	22.1	6.0	14.3	2.6
Mass balance^a	92.8	0.6	92.8	4.0

^aIncludes radioactivity recovered in cage, cage wipe and from analysis of activated charcoal trap to determine of volatile organic compounds in expired air.

Table 3. Percent recovery of radioactive dose in tissue.

	Ethyl oleate (1.7 g/kg)		Ethyl oleate (1.7 g/kg)		Ethyl oleate (3.4 g/kg)		Ethyl oleate (3.4 g/kg)
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Adrenal glands	0.01	0	0.02	0.01	0.01	0	0.01	0
Bone (both femurs)	0.05	0.03	0.03	0.01	0.03	0.02	0.02	0.01
Bone marrow (both)	0	0	0	0	0	0	0	0
Brain	0.01	0	0.01	0	0.02	0.01	0.01	0.01
Carcass (residual)	20.7	3.77	12.1	4.31	19.1	5.14	12.1	2.15
Cecum	0.1	0.03	0.08	0.03	0.11	0.02	0.08	0.05
Cecum (content/wash)	0.03	0.01	0.02	0	0.03	0.02	0.03	0.01
Colon	0.16	0.04	0.09	0.04	0.14	0.07	0.09	0.22
Colon(content/wash)	0.02	0.01	0.01	0	0.02	0.01	0.01	0
Duodenum	0.03	0.01	0.03	0.01	0.03	0.01	0.02	0.01
Duodenum(content/wash)	0.01	0.01	0.01	0	0	0.01	0.01	0
Eyes (both)	0	0	0	0	0	0	0	0
Fat (mesenteric)	0.68	0.36	0.38	0.19	0.74	0.37	0.55	0.28
Heart	0.03	0	0.03	0.01	0.02	0.01	0.02	0.01
Ileum	0.2	0.06	0.16	0.04	0.22	0.01	0.02	0.01
Ileum(content/wash)	0.05	0.01	0.05	0.02	0.06	0.01	0.07	0.01
Jejunum	0.11	0.03	0.09	0.02	0.12	0.06	0.02	0.07
Jejunum(content/wash)	0.02	0.01	0.02	0.01	0.01	0.02	0.03	0.01
Kidneys	0.12	0.02	0.1	0.02	0.12	0.03	0.1	0.01
Liver	0.51	0.06	0.52	0.15	0.45	0.09	0.4	0.12
Lungs	0.11	0.04	0.11	0.06	0.14	0.28	0.09	0.02
Lymph nodes (mes)	0.02	0	0.02	0.01	0.02	0.02	0.03	0.01
Muscles (thigh)	0.1	0.04	0.06	0.02	0.11	0.06	0.05	0.02
Ovaries	-	-	-	-	-	-	0.01	0
Pancreas	0.14	0.04	0.16	0.11	0.16	0.07	0.12	0.05
Pituitary gland	0	0	0	0	0	0	0	0
Prostate	0.02	0.01	-	-	0.02	0.01	-	-
Rectum	0.06	0.05	0.03	0.01	0.07	0.06	0.03	0.02
Salivary glands	0.04	0.01	0.03	0	0.04	0.02	0.03	0.01
Spleen	0.02	0	0.01	0.01	0.02	0.01	0.02	0.01
Stomach	0.13	0.04	0.09	0.02	0.11	0.03	0.09	0.02
Stomach(content/wash)	0	0	0	0	0	0	0	0
Testes	0.03	0.01	-	-	0.03	0.02	-	-
Thymus	0.06	0.04	0.08	0.07	0.06	0.02	0.06	0.01
Thyroid/parathyroids	0	0	0	0.01	0	0	0	0
Uterus	-	-	0.01	0	-	-	0.01	0
Total	23.2	4.34	14.4	5.06	22.1	6.03	14.3	2.56

 

Conclusions:

Under the study conditions, test substance was well absorbed with approximately 70–90% of the test substance dose absorbed. At sacrifice, the tissue with the highest concentration of radioactivity was mesenteric fat. The other organs and tissues had very low concentrations of test substance-derived radioactivity. The test substances was rapidly and extensively excreted as CO2 and approximately 40–70% of the administered dose was excreted as CO2 within the first 12 h. Fecal elimination of test substance appeared to be dose-dependent and ranged from 7% to 20% of the administered dose.

Executive summary:

A study was conducted to determine the absorption, distribution, and excretion of radiolabeled ethyl oleate in Sprague–Dawley rats after a single, peroral dose of 1.7 or 3.4 g/kg bw and was compared with a radiolabeled triacylglycerol (TG) containing only oleic acid as the fatty acid (triolein). Both test substances were well absorbed with approximately 70–90% of the test substance dose absorbed and approximately 90–100% of the TG dose absorbed. At sacrifice (72 h post-dose), tissue distribution of test substance-derived radioactivity and TG-derived radioactivity was similar. The tissue with the highest concentration of radioactivity in both groups was mesenteric fat. The other organs and tissues had very low concentrations of test substance-derived radioactivity. Both test substances were rapidly and extensively excreted as CO2 with no remarkable differences between their excretion profiles. Approximately 40–70% of the administered dose for both groups was excreted as CO2 within the first 12 h (consistent with b-oxidation of fatty acids). Fecal elimination of test substance appeared to be dose-dependent. At the dose of 1.7 g/kg, 7–8% of the administered dose was eliminated in the feces. At the dose of 3.4 g/kg, approximately 20% of the administered dose was excreted in the feces. Excretion of TG-derived radiolabel in the feces was approximately 2–4% for both doses. Overall, the results demonstrate that the absorption, distribution, and excretion of radiolabeled test substance is similar to that of TG providing evidence that the oleic acid moiety of test substance is utilized in the body as a normal dietary TG-derived fatty acid (Bookstaff, 2003).

Reference 22

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The enzymatic hydrolysis in vitro of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohols groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

other: not applicable since in vitro test

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

not applicable since in vitro test

Remarks:

100 mg of each of the substrates

No. of animals per sex per dose / concentration:

not applicable since in vitro test

Control animals:

no

Details on metabolites:

The combination of bile-pancreatic fluid digested all substrates with the exception of sorbital hexaoleate and sucrose octaoleate. Lipase (EC 3.1.1.3) did not hydrolyse compounds that contained more than three ester groups. Compounds containing four and five ester groups were hydrolysed by certain preparations of pancreatic juice; this activity is attributed to the enzyme, nonspecific lipase. This enzyme also hydrolysed esters of primary alcohols. The compounds containing six (sorbitol) and eight (sucrose) ester groups were not hydrolysed.

The rate of hydrolysis of the esters of the eight different alcohols by the various preparations of rat pancreatic juice is given in the table below. The numbers in parentheses are the volume or weight of enzyme preparation that was used in that particular digest.

Table 1. Relative rates of hydrolysis by rat pancreatic juice enzymes of the complete oleate esters of the listed alcohols.

	Pancreatic-Bile Juice	Untreated Pancreatic Juice		Treated Pancreatic Juice^a		Purified Lipase
	No TC^b	No TC	TC added	No TC	TC Added	No TC	TC added
	µmoles FFA min/ mL	µmoles FFA min/ mg		µmoles FFA min/ mg		µmoles FFA min/ mg
Methanol, 1^e	54    (0.05)^d	2.6 (1.2)	4.0 (1.2)	2.5 (1.2)	0 (1.2)	63 (0.02)	0 (0.3)
Ethylene glycol, 2	160 (0.025)	10  (0.3)	4.3 (0.3)	7.7 (0.3)	0 (0.3)	200 (0.01)	0 (0.1)
Glycerol, 3	2100 (0.005)	73 (0.075)	6.0 (0.15)	70 (0.06)	0 (0.3)	1900 (0.002)	0 (0.02)
Erythritol, 4	1.9   (1)	0   (6)	1.4 (3)	0 (6)	0 (6)	0 (0.1)	0 (0.1)
Pentaerythritol, 4	1.1   (2)	0   (6)	1.1 (3)	0 (6)	0 (6)	0 (0.1)	0 (0.1)
Adonitol, 5	0.53 (2)	0   (6)	0.25 (3)	0 (6)		0 (0.1)	0 (0.1)
Sorbitol, 6	0      (2)	0   (6)	0   (12)	0 (6)	0   (12)	0 (0.1)	0 (0.1)
Sucrose, 8	0      (2)	0   (6)	0   (12)	0 (6)	0   (12)	0 (0.1)	0 (0.1)

^a Nonspecific lipase was inactivated by treatment with α-chymotrypsin.

^b TC, Sodium taurocholate.

^c Number of ester groups.

^d The number in parentheses is the volume or weight of the enzyme preparation that was used.

The combination of bile-pancreatic fluid digested all substrates with the exception of sorbital hexaoleate and sucrose octaoleate. This failure of hydrolysis was obtained in spite of using 400 times as much combination bile-pancreatic fluid as was used when triolein was the substrate. When pancreatic juice without bile was used as a source of the enzymes, the esters that were hydrolysed depended on the presence or absence of added sodium taurocholate. In the absence of sodium taurocholate, only those substrates that contained less than four ester groups were hydrolysed. The addition of sodium taurocholate to the digest permitted the hydrolysis also of the substrates containing four and five ester groups. There were marked differences in the rates of hydrolysis of the oleate esters of methanol, ethylene glycol, and glycerol if taurocholate was not present, but these differences disappeared if this bile salt was added to the digest. In the absence of sodium taurocholate, the pattern of digestion by treated pancreatic juice. However, in the presence of added sodium taurocholate, pancreatic juice that had been treated with the proteolytic enzyme could not digest any of the substrates. The final set of results was obtained with purified pancreatic lipase. If sodium taurocholate was not present, this enzyme hydrolysed methyl oleate, ethylene glycol dioleate, and triolein, but did not hydrolyse the substrates that contained more than three ester groups. The additions of sodium taurocholate blocked completely the hydrolytic activity of this enzyme. 

Conclusions:

In the in vitro study, lipase did not hydrolyze compounds that contained more than three ester groups. However, compounds containing four and five ester groups were hydrolyzed by certain preparations of pancreatic juice; this activity is attributed to the enzyme, nonspecific lipase. This enzyme also hydrolyzed esters of primary alcohols. The compounds containing six (sorbitol) and eight (sucrose) ester groups were not hydrolyzed.

Executive summary:

An in vitro enzymatic hydrolysis of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohol groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. The combination of bile-pancreatic fluid digested all substrates with the exception of sorbital hexaoleate and sucrose octaoleate. This failure of hydrolysis was obtained in spite of using 400 times as much combination bile-pancreatic fluid as was used when triolein was the substrate. When pancreatic juice without bile was used as a source of the enzymes, the esters that were hydrolysed depended on the presence or absence of added sodium taurocholate. In the absence of sodium taurocholate, only those substrates that contained less than four ester groups were hydrolysed. The addition of sodium taurocholate to the digest permitted the hydrolysis also of the substrates containing four and five ester groups. There were marked differences in the rates of hydrolysis of the oleate esters of methanol, ethylene glycol, and glycerol if taurocholate was not present, but these differences disappeared if this bile salt was added to the digest. In the absence of sodium taurocholate, the pattern of digestion by treated pancreatic juice. However, in the presence of added sodium taurocholate, pancreatic juice that had been treated with the proteolytic enzyme could not digest any of the substrates. The final set of results was obtained with purified pancreatic lipase. If sodium taurocholate was not present, this enzyme hydrolysed methyl oleate, ethylene glycol dioleate, and triolein, but did not hydrolyse the substrates that contained more than three ester groups. The additions of sodium taurocholate blocked completely the hydrolytic activity of this enzyme (Mattson, 1972).

Description of key information

Based on the different toxicokinetic studies on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is considered to be moderately to poorly absorbed depending upon the composition. The extent of absorption of glycerides, fatty acids and fatty acids methyl esters in the gastro-intestinal system varies depending on the chain length and their degree of saturation. Generally, short-chain fatty acids are better absorbed than the long chain counterparts. Their skin permeability increases with the lipophilic nature of a compound. The tocopherols from the unsaponifiable matters were considered to be well absorbed through oral (21-86%) as well as dermal routes. However, tissue tocopherol contents tend to be related exponentially to vitamin E intake and showed no deposition or saturation thresholds. As a result, tissues vary considerably in tocopherol contents in a manner that is not related to their lipid content. Further, the sterols, sterol esters, hydrocarbons and squalene were generally poorly absorbed from the gastro-intestinal tract and slowly absorbed through the skin.

Overall, based on the toxicokinetic studies on the constituents and considering the exposed amount under the normal use conditions, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is not expected to bioaccumulate. Further, in the absence of quantitative absorption values for all the constituent types and as a conservative approach, 100% absorption rates have been considered for the hazard assessment. Additionally, for route-to-route extrapolations, default absorption factors have been used as per the ECHA guidance.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

In the absence of direct toxicokinetics data with the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters and unsaponifiable matter (including tocopherols, sterols, squalene and hydrocarbons). As the individual constituents are data rich because of their nutritional and cosmetic uses, for practical reasons, only a limited number of studies are reported below:

Glycerides:

When taken up orally, glycerides are split in the intestinal lumen into glycerol and fatty acids with the help of lipases and bile secretions, then move into enterocytes. The triglycerides are rebuilt in the enterocytes from their fragments and packaged together with cholesterol and proteins to form chylomicrons. These are excreted from the cells, collected by the lymph system and transported to the large vessels near the heart before entering the blood. Eventually, the triglycerides bind to the membranes of hepatocytes, adipocytes or muscle fibers, where they are either stored or oxidized for energy. When the body requires fatty acids as a source of energy, the hormone glucagon signals the breakdown of the triglycerides by hormone-sensitive lipases to release free fatty acids. The fatty acids are then broken down by stepwise elimination of C2-units in the mitochondrial β-oxidation. Alternate oxidation pathways can be found in the liver (ω-oxidation) and the brain (α-oxidation) (HERA, 2002).The C2 -units are esterified to acetyl-coenzyme A which directly enters the citric acid cycle where it is converted to carbon dioxide and energy (MacDonald, 1973; Robinson, 1973; Chen and Farese, 2002). Not all fatty acids present as triglycerides are used for energy production: after metabolism in the liver, redistribution to phospholipids and sterol esters may, for example, also occur (Mead and Fillerup, 1957; McArthuret al., 1999).

Glycerides with alkyl chain lengths between C8 to18, including C18-unsatd. are generally poorly water soluble have an estimated log Pow > 6 and molecular weights > 500. As such, uptake into the stratum corneum and further transfer into the epidermis are likely to be low (REACH guidance document R7.C (May 2008)).

Fatty acids:

Upon ingestion, fatty acids are directly taken up into the cells lining the intestines (enterocytes), then transported mainly in the form of triglycerides via the lymph to various tissues (see below). The fatty acids may then be stored in the form of triglycerides as a source of energy or redistributed to phospholipids and sterol esters (conversion mainly in the liver) (Mead and Fillerup, 1957; McArthur et al., 1999).

The extent of absorption in the gastro-intestinal system varies depending on the chain length of the fatty acids and their degree of saturation. Generally, short-chain fatty acids are better absorbed than the long chain counterparts. Also, absorption decreases with increasing saturation (MacDonald, 1973; Robinson, 1973; Chen and Farese, 2002). In an overview by the Cosmetic Ingredient Review Panel (CIR, 1987), stearic acid (C18) was cited as being the most poorly absorbed of the common fatty acids.

Only limited information could be located on dermal penetration of fatty acids. In dermal application studies in the rat (Butcher, 1951), linoleic acid was shown to penetrate the epithelium rapidly and reach the vascular system. Oleic acid was also reported to penetrate the epithelium of rats, possibly via hair follicles, but only minute amounts were seen in the blood vessels. Ricinoleic acid, on the other hand, was retained mainly in the outer strata of the epidermis. Other authors have noted that skin permeability increases with the lipophilic nature of a compound (Scheuplein, 1965; CIR, 1987). Dermal uptake of fatty acids has also been studied with fatty acid soaps. The C10and C12 soaps show the greatest skin penetration of human epidermis. Also, percutaneous absorption of sodium laurate is greater than that of most other anionic surfactants (HERA, 2002).

Fatty acid methyl esters:

Oral administration of radiolabeled ethyl oleate and triolein in Sprague–Dawley rats after a single, peroral dose of 1.7 or 3.4 g/kg bw were found to be well absorbed with approximately similar extent of absorption (ca. 70–90% and 90-100% absorption respectively). At sacrifice (72 h post-dose), tissue distribution of test substance-derived radioactivity and TG-derived radioactivity was similar, with maximum radioactivity found in mesenteric fat. Both test substances were rapidly and extensively excreted as CO2 with no remarkable differences between their excretion profiles. Approximately 40–70% of the administered dose for both groups were excreted as CO2 within the first 12 h (consistent with b-oxidation of fatty acids). Fecal elimination of test substance appeared to be dose-dependent, ranging from 7-20% for ethyl oleate and 2-4% for triolein (Bookstaff, 2003).

Fatty acids methyl esters are expected to be hydrolysed to the corresponding alcohol (methanol) and fatty acid by esterases (Fukami and Yokoi, 2012), even though it was shown in-vitro that the hydrolysis rate of methyl oleate was lower when compared with the hydrolysis rate of the triglyceride Glycerol trioleate (Mattson and Volpenhein, 1972). The resulting fatty acids will be metabolized via beta-oxidation, while methanol will be slowly oxidized in the liver by the enzyme alcohol dehydrogenase (ADH) to formaldehyde, which itself is oxidized very rapidly by the enzyme aldehyde dehydrogenase (ALDH) to formic acid. Finally, formic acid is slowly metabolised to CO2 and H2O (ICPS, 2002).

Little information could be found on absorption of fatty acid methyl esters via the dermal route. However, given the log Kow predictions for the representative substances, which is estimated to be >3, fatty acid methyl ester constituents are not expected to be taken up through skin to a significant degree (as per Table R 7.12 -3, REACH Endpoint specific guidance R.7c of May 2008).

Unsaponifiable matter:

Tocopherols:

The toxicokinetics of tocopherols has been extensively studied in humans, in particular due to the Vitamin E activity. α-tocopherol is the most active of all homologues, followed by β-, γ-, and δ-tocopherol. Only certain isomers are retained in human plasma, i.e. the RRR-α-tocopherol and the 2R-stereoisomers, RSR, RRS- and RSS-α-tocopherol (Traber, 1999).

Upon oral administration, α-tocopherol is absorbed unchanged from the small intestine by passive diffusion. Tocotrienol esters are first hydrolysed by pancreatic esterase (Bjørneboe et al., 1990). Absorption occurs mostly in the upper and middle thirds of the small intestine (Tomassi and Silano, 1986; Fiume, 2002) and the absorbed substance enters the lymphatic circulation (Devron, 1999). The uptake efficiency of tocopherol and its esters is generally considered to be variable. In rats given a single bolus of α-tocopherol intraduodenally, absorption was reported to be approximately 40% (Bjørneboe et al., 1990), whereas when α-tocopherol acetate was given as slow continuous infusion into the duodenum, absorption was 65% (Traber et al., 1986). In another study, the appearance of α-tocopherol in the lymph was negligible in the 2-4 h following intraduodenal dosing, reaching its peak 4-15 h after feeding (Bjørneboe et al., 1986). Intestinal absorption via the lymphatic system was 15.4%. In human studies over 24 h, absorption of α-tocopherol and its acetate ester was in the range of 21-86%. However, determination under experimental conditions may not reflect dietary reality.

α-tocopherol is rapidly transferred in plasma from chylomicrons to plasma lipoproteins, to which it binds non-specifically. The vitamin is taken up by the liver and released in low density lipoprotein (Traber et al., 1988; Combs, 1992). Most absorbed tocopherols are transported unchanged to the tissues. In non-adipose cells, vitamin E is localised almost exclusively in the membranes. Kinetic studies indicate that such tissues have two pools of the vitamin: a ’labile’, rapidly turning over pool, and a fixed, slowly turning over pool. The labile pools predominate in such tissues as plasma and liver, as the tocopherol contents of those tissues are depleted rapidly under conditions of vitamin E deprivation. In contract, the adipose vitamin E resides predominately in the bulk lipid phase, which appears to be a fixed pool of vitamin, thus, it is only slowly metabolised from this tissue (Bjørneboe et al., 1990, Combs, 1992; Basu and Dickerson, 1996). Tissue tocopherol contents tend to be related exponentially to vitamin E intake and show no deposition or saturation thresholds. As a result, tissues vary considerably in tocopherol contents in a manner that is not related to their lipid content (Machlin, 1984). Tocopherols are generally well absorbed through human skin (Fiume, 2002).

Sterols and sterol esters:

Observations in animals and humans have shown that plant sterols and sterol esters are generally poorly absorbed when taken up orally, with the highest absorption occurring for campesterol. Consumption of sterols nevertheless leads to a small but dose-related increase in plasma concentrations in short-term studies. In repeated dose rat studies, sitosterol and sitostanol were found in the adrenals, ovary and stomach at low concentrations, campestanol in the adrenals, ovaries and intestinal epithelia, and campesterol in the adrenals, spleen, intestinal epithelia, ovaries, liver and bone marrow. Excretion is via the faeces as both free sterol and sterol esters (SCF, 2003; ANZFA, 2001).

The toxicokinetics of sterol esters after oral uptake are comparable to those of sterols as they are hydrolysed to free sterols in the intestine as part of the normal digestive process (ANZFA, 2001).

No information could be found on absorption via the dermal route. However, given their high octanol/water partition coefficient (log Pow >> 8, see Section 1.3), sterols and sterol esters are not expected to be taken up through skin to a significant degree (as per Table R 7.12-3, REACH Endpoint specific guidance R.7c of May 2008).

Squalene:

Animal studies indicate that squalene is poorly absorbed from the gastro-intestinal tract and slowly absorbed through the skin. Absorption occurs through the lymphatic vessels (similar to cholesterol), with partial cyclization to sterols during the transit through the intestinal wall. Absorbed squalene is preferentially converted to bile acids in the liver (CIR, 1982).

Little information could be found on absorption via the dermal route. However, given its high log Kow >> 6, squalene is not expected to be taken up through skin to a significant degree (as per Table R 7.12 -3, REACH Endpoint specific guidance R.7c of May 2008). This is in line with what was seen in a study by Butcher (1951) who saw little evidence that squalene penetrated through the skin of rats when applied dermally.

Hydrocarbons:

Absorption of alkanes may occur through the portal and/or the lymphatic system. For n- and cycloalkanes the oral absorption varies from 90% for C14-C18 to 25% for C26-C29. The absorption further decreases with increasing carbon number, until above C35 when it is negligible. Limited data suggest that cyclo-alkanes are absorbed at similar levels as n-alkanes of comparable molecular weight, whereas absorption of branched alkanes is slightly less (EFSA, 2012). Alkanes are initially oxidised to the corresponding fatty alcohols by the cytochrome P450 system, subsequently biotransformed to fatty acids and in some cases subjected to the normal β-oxidation pathway. This reaction is more rapid for n-alkanes than for branched- and cyclo-alkanes. Due to low biotransformation rates, in particular for some branched- and cyclo-alkanes, Mineral oil saturated hydrocarbons (MOSH) having carbon number between 16 and 35 may accumulate in different tissues including adipose tissue, lymph nodes, spleen and liver. In rats, the terminal half-life of MOSH in blood (estimated from P15(H) white oils) was between 23 and 59 h, depending on the strain. However, this reflects the elimination of the easily degraded MOSH. The concentration of MOSH in human tissues (mainly lymph nodes, liver, spleen and adipose tissue) demonstrates that accumulation of these compounds, mostly branched- and cyclo-alkanes, occurs in humans (EFSA, 2012).

With regard to the dermal route, studies with humans, animals, and excised skin have all shown that C5–C12 alkanes are poorly absorbed following dermal administration (Mckee et al., 2015).