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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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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)
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
- Source: Victorian Chemical, Victoria, Richmond, Australia
- Analytical purity: 80.6 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as given in publication: Sprague–Dawley rats [Crl:CD(SD)IGS BR]
- 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: AIN-93G 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:
The test item was formulated into the diet. For this purpose, 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 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.
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:
3.3, 6.7 and 10%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Females: 2.0, 3.9, 6.1 g/kg bw/day
Basis:
other: mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3, 6.7 and 10 % in feed.
Remarks:
Doses / Concentrations:
Males: 1.8, 3.6, 5.5 g/kg bw/day
Basis:
other: mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3, 6.7 and 10 % in feed
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle

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.

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

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined weekly. Compound intake (g/kg body weight/day) was calculated from actual body weight and food consumption data and target concentration of ethyl oleate in feed.

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 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, hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, leucocyte count, differential blood cell count, blood smear, prothrombin time (PTT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes, see hematology
- Parameters checked: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, alkaline phosphate (AP), gamma-glutamyl transferase (gamma-GT), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), calcium, inorganic phosphorus, sodium, cholesterol, total bilirubin, potassium, chloride, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: urine was collected overnight 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: all groups, 10 animals/sex/group
Testings performed:
- Hand-held and open-field observations (10 animals/sex/group 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 (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 (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.

PLASMA ETHYL OLEATE CONCENTRATION MEASUREMENTS
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 above), 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: all animals were subjected to gross pathological examination
ORGAN WEIGHTS: 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, epididymides (2), spleen, heart, testis (2), kidney (2), thymus, liver, thyroid with parathyroid, ovary (2), uterus.
HISTOPATHOLOGY: histopathology was done in control and high dose group animals.
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.
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
Other examinations:
Spermatocyte assessment in males and evaluation of the estrous cycle in females.
Statistics:
Control versus treated group comparisons 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):
decreased absolute body weight and body weight gain, 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):
some changes 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):
some changes considered non-adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
some findings 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 low dose group male was found dead on day 39, and two mid dose group males were found dead on days 30 and 93. These unscheduled deaths were unrelated to the test item.

BODY WEIGHT AND WEIGHT GAIN
Both absolute terminal body weight and body weight gains of the mid and high dose group females were statistically significantly lower than the control group. Absolute body weights were 90.8 and 90.5% of the control group for the mid dose and the high dose females, respectively. This finding did 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 was directly related to lower food consumption relative to the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE
The lower food consumption relative to controls was fully consistent with a decrease in the palatability of the EO-containing food versus the triglyceride-containing food. In fact, a decrease in food consumption was noted within the first week (consistent with palatability preferences) and there was no dose-response relationship with regard to food consumption, since mid dose animals consumed less than high dose; furthermore, no cumulative decreases in food consumption as often observed in case of toxicity was here noticed. Moreover, anecdotal experience data from the testing facility showed that rats prefer diets containing high triglyceride fat over high EO-fat.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related findings.

HAEMATOLOGY
There were no treatment-related changes in parameters.

CLINICAL CHEMISTRY
The only statistically significant differences after 13 weeks of treatment were minimally lower calcium and mildly lower inorganic phosphorus levels for males fed diets containing 10% EO (high dose group). 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 behaviour 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 mid and high dose group females vs. control, and a slight increase in brain-to-body weight percent in the mid and high dose group females, which was driven by the decrease in terminal body weight seen in these groups.

GROSS PATHOLOGY
No gross abnormalities were reported.

HISTOPATHOLOGY
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 represented small amounts of unabsorbed EO at the mid- and high-dose levels (estimates of EO absorption in this study were >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 the low dose group at day 60 (0.024 μg/mL), and the other was a control 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