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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Due to similar chemical structures except for the esterified alcohol moiety the toxicokinetics of the test material propyl oleate is expected to be comparable to the toxicoknietic properties of ethyl oleate under the reported conditions.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2003

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
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).
Deviations:
not specified
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Ethyl oleate was obtained from Victorian Chemical, Victoria, Richmond, Australia. Analytical testing showed it complied with both National Formulary (NF) and European Pharmacopoeia (EP) specifications. The EO used in this study is oil derived from the ethylation of sunflower oil. To be compliant with the NF and EP monographs for EO, the fraction of the oil that is actually the EO molecule must be at least 60%. The EO used in this study was analyzed by GC to determine fatty acid composition, and by HPLC to determine exact concentration of EO (determined to be 80.6%).
Specific details on test material used for the study:
- Name of test material (as cited in study report): (14C) Ethyl oleate
- Specific activity (if radiolabelling):165 µCi/mg
- Radiochemical purity (if radiolabelling): HPLC analyses showed the mean radiopurity of (14C) Ethyl oleate to be 99.0%
- Locations of the label (if radiolabelling): The radiolabel was on the carboxyl carbon.
- Other: (1-14C)Ethyl oleate was synthesised via the esterification of (1-14C) oleic acid with thionyl chloride in the presence of ethanol.
The radiolabeled compound was formulated as a 34% w/w lipid emulsion for dosing (non-radiolabeled ethyl oleate was used as the carrier for the labeled ethyl oleate).
Radiolabelling:
yes
Remarks:
(14C) Ethyl oleate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley [Crl:CD (SD) IGS BR]
Sex:
male/female
Details on test animals 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)
Details on exposure:
single doses: 1.7 or 3.4 g/kg
Duration and frequency of treatment / exposure:
single doses
Doses / concentrationsopen allclose all
Dose / conc.:
1 700 mg/kg bw/day (nominal)
Remarks:
Each animal recieved approximately 120 µCi/mg of radioactivity.
Dose / conc.:
3 400 mg/kg bw/day (nominal)
Remarks:
Each animal recieved approximately 120 µCi/mg of radioactivity.
No. of animals per sex per dose:
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

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)ethyl oleate. 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 ethyl oleate 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) ethyl oleate- 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 for the lower (1.7 g/kg) dose levels below.
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) ethyl oleate, 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 ethyl oleate-derived radioactivity was similar to that of triacylglycerol (TG)-derived radioactivity. Again, this supports the conclusion that ethyl oleate 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 ethyl oleate-derived radioactivity and triacylglycerol-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

Results

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 ethyl oleate (EO) and other fatty acid esters are rapidly hydrolysed to ethanol and free fatty acid within the GI tract. 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 ethyl oleate and the triacylglycerol groups indicating that both lipids deliver free fatty acid to tissue for oxidation at approximately the same rate and extent.

Table 1: 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 2: 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.

  Percent of dose
  (14C) Ethyl oleate (1.7 g/kg)
   Males     Females   
   Mean (n=5)     Mean (n = 5)   
 Urine        
 0 - 24  1.62  0.10  1.84  0.13
 24 - 48  1.88  0.11  2.09  0.14
 48 - 72  1.98  0.12  2.21  0.13
 Feces        
 0 - 24  6.81  1.83  7.57  1.25
 24 - 48  7.50  1.80  8.24  1.10
 48 - 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

 Tissue

 

 

 

 

 72 h

 23.2

 4.3

 14.4

 5.1

 Mass balance*

 95.3

 2.0

 95.9

 0.7

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

At the lower dose level (1.7 g/kg), distribution of radioactivity into tissues was similar between [14 C]ethyl oleate and [14 C]triacylglycerol-treated rats, which suggests that ethyl oleate is hydrolyzed, absorbed, resynthesized into triacylglycerol, and distributed similar to dietary sources of oleic acid. At the higher dose level, the percentage of radioactivity that accumulated into tissue was higher in the triacylglycerol-treated rats than in the ethyl oleate-treated rats, probably due to the decreased absorption of [14 C]EO-derived radioactivity at the higher dose level. In all groups, a very high percentage of the radioactivity recovered from tissues was associated with the residual carcass (84–89%), with males in all groups trending slightly higher than the corresponding females. The carcass can be thought to primarily consist of muscle, skin and fat. The high percentage of radioactivity found in the carcass can be attributed to the relatively high mass of the carcass relative to other tissues, as well as the presence of fat in the carcass. The tissue to plasma ratio of radioactivity of [14 C]ethyl oleate at both doses was either similar or lower for all tissues collected, when compared to corresponding treatments with [14 C]triacylglyceride, indicating that [14 C]ethyl oelate-derived radioactivity is distributed in a manner similar to triacylglyceride. The principal route of excretion of radioactivity for both [14 C]ethyl oleate and [14 C]triacylglyceride was via expired air. Excretion of [14 C]CO2 was rapid in all groups such that by 12 h after dosing 40–70% of the administered dose was excreted in expired air. The generation of CO2 results from the process of fatty acid oxidation, termed beta-oxidation.

Applicant's summary and conclusion

Conclusions:
Ethyl oleate is rapidly and extensively hydrolyzed to free oleic acid, absorbed, and delivered to tissues where it undergoes beta-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 ethyl oleate-derived radioactivity was similar to that of Triglyceride-derived radioactivity. Again, this supports the conclusion that ethyl oleate is rapidly hydrolyzed to oleic acid, absorbed, and distributed within the body in the same way as dietary sources of oleic acid. The similar tissue distribution between ethyl oleate-derived radioactivity and triglyceride-derived radioactivity suggests that the radiolabel in tissue represents the same chemical form (i.e., the oleic acid moiety). Due to similar chemical structures except for the esterified alcohol moiety the toxicokinetics of the test material propyl oleate is expected to be comparable to the toxicoknietic properties of ethyl oleate under the reported conditions.
Executive summary:

Ethyl oleate is rapidly and extensively hydrolyzed to free oleic acid, absorbed, and delivered to tissues where it undergoes beta-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 ethyl oleate-derived radioactivity was similar to that of triglyceride-derived radioactivity. Again, this supports the conclusion that ethyl oleate is rapidly hydrolyzed to oleic acid, absorbed, and distributed within the body in the same way as dietary sources of oleic acid. The similar tissue distribution between ethyl oleate-derived radioactivity and triglyceride-derived radioactivity suggests that the radiolabel in tissue represents the same chemical form (i.e., the oleic acid moiety). At the high dose (3.4 g/kg), there is decreased absorption of ethyl oleate-derived radioactivity as shown by increased fecal elimination. This appears to be the most significant difference between ethyl oleate and triglyceride and indicates that the capacity and/or rate of hydrolysis are different between ethyl oleate and triglyceride. The decreased absorption of ethyl oleate at the high dose of 3.4 g/kg results in lower tissue concentrations of ethyl oleate-derived radioactivity than triglyceride-derived radioactivity. However, relative tissue distribution of ethyl oleate-derived radioactivity remained similar to triglyceride-derived radioactivity. In the human safety study, there were no clinically significant negative effects from consuming EO at levels up to 16 g/day (approximately 0.2 g/kg). Due to similar chemical structures except for the esterified alcohol moiety the toxicokinetics of the test material propyl oleate is expected to be comparable to the toxicoknietic properties of ethyl oleate under the reported conditions.