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

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

The safety of the use of ethyl oleate in food is supported by metabolism data in rats and clinical safety data in humans

Author:

Bookstaff RC et al

Year:

2003

Bibliographic source:

Regul Toxicol Pharmacol. 2003 Feb;37(1):133-48

Materials and methods

Objective of study:

other: ADME

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl oleate

EC Number:

203-889-5

EC Name:

Ethyl oleate

Cas Number:

111-62-6

Molecular formula:

C20H38O2

IUPAC Name:

ethyl (Z)-octadec-9-enoate

Test material form:

not specified

Radiolabelling:

yes

Remarks:

(14C) Ethyl oleate

Test animals

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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

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

Results and discussion

Main ADME results

Type:

absorption

Results:

75-88%

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

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).

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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).