Propyl oleate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Analogue approach: read-across from a single source substance to a single structurally similar target substance (see read across document for details).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: FDA draft ‘‘Redbook II’’ guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, 1993)

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents

Deviations:

not specified

Principles of method if other than guideline:

The purpose of this study was to determine the safety of ethyl oleate (EO) in a 91-day feeding study in Sprague–Dawley rats.

GLP compliance:

yes

Test type:

other: 91-day feeding study

Limit test:

no

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 octadec-9-enoate

Constituent 2

Reference substance name:

Ethyl stearate

EC Number:

203-887-4

EC Name:

Ethyl stearate

Cas Number:

111-61-5

Molecular formula:

C20H40O2

IUPAC Name:

ethyl stearate

Constituent 3

Reference substance name:

Ethyl palmitate

EC Number:

211-064-6

EC Name:

Ethyl palmitate

Cas Number:

628-97-7

Molecular formula:

C18H36O2

IUPAC Name:

ethyl palmitate

Constituent 4

Reference substance name:

Ethyl linoleate

EC Number:

208-868-4

EC Name:

Ethyl linoleate

Cas Number:

544-35-4

Molecular formula:

C20H36O2

IUPAC Name:

ethyl octadeca-9,12-dienoate

Constituent 5

Reference substance name:

Ethyl docosanoate

EC Number:

227-616-4

EC Name:

Ethyl docosanoate

Cas Number:

5908-87-2

Molecular formula:

C24H48O2

IUPAC Name:

ethyl docosanoate

Constituent 6

Reference substance name:

Eicosanoic acid, ethyl ester

Cas Number:

18281-05-5

Molecular formula:

C22H44O2

IUPAC Name:

Eicosanoic acid, ethyl ester

Constituent 7

Reference substance name:

ethyl (Z)-icos-9-enoate

Cas Number:

393803-94-6

Molecular formula:

C22H42O2

IUPAC Name:

ethyl (Z)-icos-9-enoate

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:

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 ethyl oleate used in this study is oil derived from the ethylation of sunflower oil. To be compliant with the NF and EP monographs for ethyl oleate, 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 ethyl oleate (determined to be 80.6%).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals and environmental conditions: Sprague–Dawley rats [Crl:CD(SD)IGS BR] were obtained from Charles River Laboratory. This strain of rats is commonly used for this type of study and there is considerable historical data on this strain of rats. At dosing, the animals weighed approximately 150–175 g and were approximately 5–6 weeks of age. Rats were housed individually in stainless-steel cages. Temperature and humidity were controlled throughout the duration of the study. Diet and water were available, ad libitum, throughout the study period, with the exception of the night prior to blood collections for which animals were fasted. Rats were divided into four dose groups of 20 rats per gender.

Diet: Test materials were formulated into a purified diet based on the AIN-93G purified diet. The AIN-93G diet was modified to allow incorporation of an additional 10% of test fat (either Ethyl oleate, High oleic safflower oil (HOSO), or a combination of the
two). The modification involved decreasing overall carbohydrate concentration to allow the incorporation of an additional 10% of test fat without diluting out other nutrients.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Test material stability:

Data on test material stability, homogeneity, and diet concentrations shows that the test material was stable over the course of the study, and diet concentrations
were homogeneous and within 10% of target.

Diet:

Test materials were formulated into a purified diet based on the AIN-93G purified diet. The AIN-93G diet was modified to allow incorporation of an additional 10% of test fat (either EO, HOSO, or a combination of the two). The modification involved decreasing overall carbohydrate concentration to allow the incorporation of an additional 10% of test fat without diluting out other nutrients.

Doses:

1. Control 0 % ethyl oleate; 10 % high oleic safflower oil
2. Ethyloleate Low 3.3 % ethyl oleate, 6.7 % high oleic safflower oil
3. Ethyloleate Mid 6.7 % ethyl oleate, 3.3 high oleic safflower oil
4. Ethyloleate High 10 % ethyl oleate, 0 % high oleic safflower oil

The diet also contained soybean oil providing 5% fat in all groups.

No. of animals per sex per dose:

20 male and 20 female rats per dose

Control animals:

yes

Details on study design:

91-day feeding study according to 1993 FDA draft ‘‘Redbook II’’ guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food). The study was conducted under Good Laboratory Practices (GLP) at Covance Laboratories (Madison, WI). Sprague–Dawley rats [Crl:CD(SD)IGS BR] were obtained from Charles River Laboratory. Expanded clinical observations were performed once prior to treatment, and then weekly throughout the study as described below.

Statistics:

Control versus treated group comparisons (Groups 2 through 4 versus Group 1) were evaluated at the 5.0%, two-tailed probability level. Data for each sex were analyzed separately. If Levene's test for variance homogeneity was not significant (p > 0:05), one-way analysis of variance (ANOVA) was performed on the observed values. If Levene's test was significant (p < 0:05), ANOVA was done on the rank transformed data. Post-hoc Dunnett's t-test was used for control versus treated group mean comparison, incorporating transformations when necessary.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

>= 2 000 mg/kg bw

Based on:

test mat. (total fraction)

Mortality:

Three rats died during the course of the study: A group 2 male was found dead on day 39, and two group 3 males were found dead on days 30 and 93. Gross
pathology observations on the group 2 male showed moist yellow staining in the perineum/perianal region, red nasal discharge, and urogenital findings including
enlarged kidney and thickened dark red bladder wall containing multiple tan and firm calculi (up to 10 mm in diameter). Findings on the group 3 males were less definitive and included tan and creamy semifluid in the stomach and enlarged pelvis (day 30 rat). There were no microscopic correlates in these animals. There were no macroscopic or microscopic lesions reported for the rat found on day 93. These unscheduled deaths were judged by the Pathologist to be unrelated to the test material.

Clinical signs:

The appearance, general condition, and behavior of the rats was not affected by ethyl oleate at any dose level. Findings were either common to all groups and sexes or they were incidental in nature. Likewise, there were no visible changes in the feces of the rats.

Body weight:

Both absolute terminal body weight and body weight gains of the group 3 and 4 females were statistically significantly lower than the control group. Absolute body weights were 90.8 and 90.5% of the control group for groups 3 and 4 females, respectively. Mean consumption of ethyl oleate in the high-dose group over the course of the study was 5.5 (males) and 6.1 g/kg/day (females).

Gross pathology:

The only statistically significant differences present after 13 weeks of treatment were minimally lower calcium and mildly lower inorganic phosphorus for males fed diets containing 10% ethyl oleate. There were no correlative findings for these minor differences, and they were not considered adverse or toxicologically meaningful. There were no treatment-related effects in any of the urinalysis parameters at any time.

Any other information on results incl. tables

Results

Plasma concentration of ethyl oleate:

The plasma concentration of ethyl oleate was measured following an overnight fast at 1 month, 2 months, and 3 months. Only two rats had quantifiable levels (above 0.02 µg/ml). One was a female rat in Group 2 at Day 60 (0.024 µg/ml), and the other was a Group 1, female at termination (0.06 µg/ml of ethyl oleate). There were no other rats with EO measurements greater than 0.02 µg/ml of ethyl oleate.

Histopathology:

Histopathology was conducted on control and high-dose animals. Hepatocellular vacuolation typical of fat accumulation was noted for both control and high-dose animals. The incidence and severity of the vacuolation were higher for animals given 10% HOSO (controls) than for the animals given 10% ethyl oleate. Evaluation of other organs/tissues did not reveal any test article-related findings.

Organ weights

The only statistically significant effects were a decrease in terminal body weight in the Group 3 and 4 females vs. Group 1, and a slight increase in brain-to-body weight percent in the Group 3 and 4 females,

which is driven by the decrease in terminal body weight seen in these groups.

Ophthalmic observations

There were no treatment-related ophthalmic findings.

Discussion (excerpt)

The study produced no evidence of toxicity in rats that consumed ethyl oleate at up to 10% of their diet for at least 91 days. The study was conducted according to the robust guidelines put forth by the FDA in their publication: Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, 1993 (commonly referred to as Redbook II). With regard to the standard parameters measured in a subchronic toxicity study, there were few statistically significant findings. The terminal body weights of the group 3 and 4 females were statistically-significantly lower (90.8 and 90.5% of the control group, respectively). This finding was not considered to be a toxic response since the body weight gain of these two groups over the course of the study (160 g, each) is greater than

the 144 g normally seen in this strain of rats, in this laboratory, during a similar time period, within a dietary study (Historical Control In-Life Data, Covance Laboratories-Madison, 1998–2001).

The lower body weight relative to control rats is directly related to lower food consumption relative to the controls. The lower food consumption relative to controls is fully consistent with a decrease in the palatability of the ethyl oleate-containing food versus the triglyceride-containing food. This conclusion is based on (1) lower food consumption was noted within the first week (consistent with palatability preferences), (2) there was not a doseresponse with regard to food consumption (mid-dose consumed less than high-dose), (3) the lack of cumulative decreases in food consumption which often are

observed with toxicity, and (4) anecdotal experiences in our lab show that rats prefer diets containing high triglyceride fat over high ethyl oleate fat. The relationship between palatability and reduced

food consumption has been recently addressed (Mayhew et al., 2003). As palatability decreases, food consumption decreases. This paper shows that in the absence of toxicity, reduced palatability leads to a small, but constant decrease in food consumption that is not dose-related. This relationship is what was seen in the present study.

Conclusion

In conclusion, the data generated in this study show that consumption of ethyl oleate in the diet of rats at up to 10% of the diet for 91 days (consumption generally ranged between 4 and 8 g ethyl oleate/kg body weight/day) was well-tolerated and devoid of any toxic effects. The No Observable Effect Level (NOAEL) was determined to be 6.7% ethyl oleate in the diet (based on the small decreases in calcium and phosphorous in the high-dose males). The No Observable Adverse Effect Level was determined to be 10% EO in the diet based on the lack of any clear adverse effects seen in this study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the data generated in this study show that consumption of ethyl oleate in the diet of rats at up to 10% of the diet for 91 days (consumption generally ranged between 4 and 8 g ethyl oleate/kg body weight/day) was well-tolerated and devoid of any toxic effects. Based on the conclusion of the 91 d feeding study in rats the LD50 after single oral application in rats of ethyl oleate is > 2000 mg/kg bw.

Executive summary:

In conclusion, the data generated in this study show that consumption of ethyl oleate in the diet of rats at up to 10% of the diet for 91 days (consumption generally ranged between 4 and 8 g ethyl oleate/kg body weight/day) was well-tolerated and devoid of any toxic effects. The No Observable Effect Level (NOAEL) was determined to be 6.7% ethyl oleate in the diet (based on the small decreases in calcium and phosphorous in the high-dose males). The No Observable Adverse Effect Level was determined to be 10% EO in the diet based on the lack of any clear adverse effects seen in this study.