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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented published GLP Guideline study.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: 1993 FDA draft "Redbook II" guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
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
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyl oleate (EO); 9-octadecenoic acid ethyl ester
- CAS No. of test material (as cited in study report): 111-62-6
- Source: Victorian Chemical, Victoria, Richmond, Australia
- Analytical purity: 80.6 %
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%). Table 1 shows the fatty acid composition (as percent of total fatty acids) of both the EO oil and the HOSO, for those fatty acids 0.1% or greater. For EO, the fatty acids are in the form of ethyl esters and for HOSO, the fatty acids are as triglycerides.

Test animals

Species:
rat
Strain:
other: Sprague–Dawley rats [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: approx. 5-6 weeks
- Weight at study initiation: approx. 150–175 g
- Fasting period before study: one night prior to blood collections
- Housing: individually in stainless steel cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
Temperature and humidity were controlled throughout the duration of the study.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet containing high oleic safflower oil (HOSO)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
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. The composition of this basal diet is shown in Table 2.

In this study, the test diets were prepared based on the addition of the EO oil (i.e., the high-dose diets contained 10% of the EO oil), and were not adjusted based on the actual concentration of the EO molecule.

VEHICLE
- Source: High oleic safflower oil (HOSO) was obtained from Columbus Foods Company, Chicago, Illinois.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on test material stability, homogeneity, and diet concentrations showed that the test material was stable over the course of the study, and diet concentrations were homogeneous and within 10% of target (data not shown).
Duration of treatment / exposure:
91 days
Frequency of treatment:
daily ad libitum feeding
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2.0, 3.9, 6.1 g/kg bw/d
Basis:
other: females, calculated from actual body weight and food consumption data and target concentration of ethyl oleate in feed
Remarks:
Doses / Concentrations:
0, 1.8, 3.6, 5.5 g/kg/day
Basis:
other: males, calculated from actual body weight and food consumption data and target concentration of ethyl oleate in feed
Remarks:
Doses / Concentrations:
0, 3.3, 6.7, and 10%
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
EO was mixed into AIN-93G purified diet at levels of 0, 3.3, 6.7, and 10% by weight (the high-dose males and females consumed 5.5 and 6.1g/kg/day EO, respectively). All diets were calorie- and fat-matched using high oleic safflower oil (HOSO) as the control fat.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, outside the home cage and included changes in skin, fur, eyes, mucous membranes, occurrences of secretions and excretions, changes in posture, and reactivity to handling. Changes in gait were assessed weekly by allowing the animal to walk freely to allow evaluation of gait.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were taken pre-study for randomization, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
Food consumption was determined weekly.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once prior to treatment and once during Week 13
- Dose groups that were examined: all animals were examined in random order by a boardcertified veterinary ophthalmologist using an indirect
ophthalmoscope and a slit lamp. The eyes were dilated with a mydriatic agent prior to examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Following an overnight fast, blood was taken (unanesthetized) from 10 animals/sex/group on Days 30, 60,
and all animals at scheduled sacrifice. The same 10 animals/sex/group were used for the Days 30 and 60 collections. Blood was collected via the jugular vein.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked: erythrocyte count, Hb, Hct, MCV, MCH, MCHC, platelets, leucocyte count, differential blood cell count, blood smear, PTT, activated PTT

CLINICAL CHEMISTRY: Yes, see hematology
- Parameters checked: Glucose, urea nitrogen, cretinine, total protein, albumin, globulin, AP, gamma-GT, ASAT, ALAT, Calcium, inorganic phoshorus, sodium, cholesterol, total bilirubin, potassium, chloride, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected overnight in containers on wet ice before blood collection.
- Animals fasted: Yes
- Parameters checked: appearance, bilirubin, blood, glucose, ketones, microscopic examination of sediment, pH, protein, specific gravity, urobilinogen, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once prior to treatment, and then weekly throughout the study
- Dose groups that were examined: 10 animals/sex/group
Testings performed:
- Hand-held and open-field observations (10 animals/sex/group (chosen by random number) prior to treatment and weekly during the study): Reactivity to handling, vocalization, palpebral closure, exophthalmos, excessive lacrimation, excessive salivation, respiration, appearance of fur, piloerection, muscle tone, and pupillary status.
- Elicited behaviors observations (10 animals/sex/group (the same 10 animals/sex/group as those used for hand-held and open-field observations) once during Week 13: Auditory reactivity, proprioceptive positioning reaction, pinna response, pupillary status, pupillary response, grip strength, and nociceptive reflex
- Motor activity (10 animals/sex/group (again, the same 10 animals/sex/group as those used above) once during Week 13): The animals were placed into an automated photocell activity-recording device and activity was recorded for 40 min.

ORGAN WEIGHTS
At sacrifice, the following organs were weighed (paired organs weighed together). Organ-to-body weight percentages and organ-to-brain weight ratios were calculated: Adrenal (2), Pituitary gland, Brain, Prostate, Epididymis (2), Spleen, Heart, Testis (2), Kidney (2), Thymus, Liver, Thyroid with parathyroid, Ovary (2), Uterus

PLASMA ETHYL OLEATE CONCENTRATION MEASUREMENTS
Following an overnight fast, blood was taken from 10 animals/sex/group on Days 30, 60, and all animals at scheduled sacrifice for the purpose of measuring plasma concentrations of EO. The same 10 animals/sex/group were used for the Days 30 and 60 clinical pathology blood collections. Approximately 220 μL of blood collected from the jugular vein was placed into a Microtainer tube (containing 30 μL of a 3.8% sodium citrate solution) and was gently and thoroughly mixed on a vortex. Duplicate aliquots (50 μL each) were then immediately transferred using calibrated, positive displacement pipettes into prepared Sarstedt PP microvials (duplicate) containing 50 μL of stable isotopically labeled internal standard (SILIS) prepared solution and 1ml of acetone. The vials were gently mixed on a vortex mixer and placed on ice. Disposition/mixing of blood into prepared vials was completed within 4 min from the time that the blood was collected from the animal. This process stabilizes EO, preventing further hydrolysis. Samples were frozen until analyzed.
Plasma samples were analyzed for EO using LC/MS/MS with atmospheric pressure chemical ionization in the positive ion mode using selective reaction monitoring. The method's calibration range was 0.02–10 μg/mL.

FECAL FAT EVALUATION
Feces were collected from 10 animals/sex/group on Days 29 and 59 (animals that were selected for clinical pathology tests), and all animals on Day 89. The same 10 animals/sex/group were used for the Days 29 and 59 collections. Feces were collected overnight (animals were not fasted) from the cage-pan. The fecal material was sifted in a fine-grade sifter to remove loose residual feed, weighed for each animal, and stored in a freezer set to maintain -60º to -80º C until analyzed for total fat content using AOAC Method No. 95402.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all
HISTOPATHOLOGY: Yes, control and high dose group
After 13 weeks of treatment, all surviving animals were fasted overnight, bled for clinical pathology tests and for the special blood collection sample, anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied.
The following tissues (when present) from each animal, with the exception of testes, were preserved in 10% neutral-buffered formalin and slides prepared for histopathological examination. Testes were preserved in Bouins fixative. Organs from animals in the control and high-dose groups were examined histopathologically:
Adrenal (2)
Aorta
Brain (cerebrum, cerebellum, and medulla)
Cecum
Cervix
Colon [proximal and distal (2)]
Duodenum
Epididymis (2)
Esophagus
Eye (2)
Femur with bone marrow (articular surface of
the distal end)
Harderian gland
Heart
Ileum (including Peyers patch)
Jejunum
Kidney (2)
Lacrimal gland (exorbital)
Liver
Lung with mainstem bronchi
Lymph node (mandibular and mesenteric)
Mammary gland (females)
Nasal turbinates
Ovary (2)
Pancreas
Pituitary gland
Prostate
Rectum
Salivary gland [mandibular (2)]
Sciatic nerve
Seminal vesicle (2)
Skeletal muscle (thigh)
Skin
Spinal cord (cervical, thracic, lumbar)
Spleen
Sternum with bone marrow
Stomach (nonglandular and glandular)
Testis [preserved in Bouins fixative for
sacrificed animals (2)]
Thymus
Thyroid with parathyroid
Tissues with macroscopic changes or alterations
(i.e., gross lesions)
Tongue
Trachea
Urinary bladder
Uterus with uterine horns
Vagina
Zymbals gland
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

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
considered non-adverse
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased food intake due to lower palatability
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
considered non-adverse
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
considered non-adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
considered non-adverse
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
The appearance and general condition of the rats was not affected by EO 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. 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. These unscheduled deaths were judged by the Pathologist to be unrelated to the test material.

BODY WEIGHT AND WEIGHT GAIN
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. This finding does not represent a toxicologically significant effect because rats on the EO diets gained more weight during the course of the study than historical control data on this strain of rats. The lower body weight relative to control rats is directly related to lower food consumption relative to the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The lower body weight relative to control rats was 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 EO-containing food versus the triglyceride-containing food. This conclusion is based on (1) a decrease in food consumption was noted within the first week (consistent with palatability preferences), (2) there was not a dose-response 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 EO-fat.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmic findings

HAEMATOLOGY
There were no treatment-related effects

CLINICAL CHEMISTRY
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% EO. There were no correlative findings for these minor differences, and they were not considered adverse or toxicologically meaningful.

URINALYSIS
There were no treatment-related effects in any of the urinalysis parameters at any time.

NEUROBEHAVIOUR
The behavior of the rats was not affected by EO at any dose level.

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.

GROSS PATHOLOGY
no data

HISTOPATHOLOGY: NON-NEOPLASTIC
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% EO. Evaluation of other organs /tissues did not reveal any test
article-related findings.

FECAL FAT CONCENTRATION
There was a dose-related increase in fecal fat concentration in both sexes from approximately 9% (control) to 18% in males, and from 4 (control) to 13% in females There were no visually obvious differences with regard to feces quality or quantity at any level of EO in the diet (i.e., color, diarrhea, weight, etc.). The increase in fat most likely represents small amounts of unabsorbed EO at the mid- and high-dose (estimates of EO absorption in this study are >80%).

PLASMA EO CONCENTRATION MEASUREMENTS
The plasma concentration of EO 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 EO). There were no other rats with EO measurements greater than 0.02 μg/mL of EO.

Effect levels

Dose descriptor:
NOAEL
Effect level:
5 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical observations, body weight gains, appearance of the feces, ophthalmic examinations, hematology, clinical chemistry, urinalysis, organ weights, histopathology, or male and female reproductive assessments

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion