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Description of key information

Oral (OECD 408), 90 days, rat: NOAEL (systemic) ≥ 3460 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented, published GLP guideline study.
Qualifier:
according to guideline
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
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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.
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
Dose / conc.:
3.3 other: % (nominal in diet)
Remarks:
Females: 2.0 g/kg bw/day / Males: 1.8 g/kg bw/day (mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3% in feed)
Dose / conc.:
6.7 other: % (nominal in diet)
Remarks:
Females: 3.9 g/kg bw/day / Males: 3.6 g/kg bw/day (mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 6.7% in feed)
Dose / conc.:
10 other: % (nominal in diet)
Remarks:
Females: 6.1 g/kg bw/day / Males: 5.5 g/kg bw/day (mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 10% in feed)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
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.
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.
Key result
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
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 Jun - 08 Dec 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No details on analytical purity. No detailed clinical observations and no ophthalmoscopic and neurobehavioural examinations were performed. Not all required parameters were examined at clinical chemistry and haematology analysis.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
no details on analytical purity; no detailed clinical observations and no ophthalmoscopic and neurobehavioural examinations were performed; not all required parameters were examined at clinical chemistry and haematology analysis
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Tac. N(SD) fBR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Germantown, N.Y., USA
- Age at study initiation: 9 weeks (males), 10 weeks (females)
- Weight at study initiation: 215-305 g (males), 179-233 g (females)
- Housing: animals were housed individually in stainless steels wire floored cages
- Diet: Purina Laboratory Chow #5001, ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, not further specified
- Humidity (%): controlled, not further specified
- Photoperiod (hrs dark / hrs light): controlled, not further specified

IN-LIFE DATES: From: 15 Jun 1982 To: 16 Sept 1982
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks (93 days)
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
1 other: mL/kg bw (actual ingested)
Remarks:
865 mg/kg bw/day (actual ingested; calculated based on a density of 0.865 g/mL)
Dose / conc.:
2 other: mL/kg bw (actual ingested)
Remarks:
1730 mg/kg bw/day (actual ingested; calculated based on a density of 0.865 g/mL)
Dose / conc.:
4 other: mL/kg bw (actual ingested)
Remarks:
3460 mg/kg bw/day (actual ingested; calculated based on a density of 0.865 g/mL)
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The doses selected in this study were based on a previous range-finding study in rats.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed once daily for general appearance and behaviour and twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: body weights and body weight gains were determined weekly during the study period.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood was drawn via orbital sinus puncture at Week 7 and 13, respectively.
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes, overnight (16 h prior to blood sampling)
- How many animals: all animals
- Parameters checked: haemoglobin (Hb), haematocrit (HCT), red blood cell count (RBC), total and differential white blood cell count (WBC), mean cell volume (MCV), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood was drawn via orbital sinus puncture at Week 7 and 13, respectively.
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes, overnight (16 h prior to blood sampling)
- How many animals: all animals
- Parameters checked: serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), serum alkaline phosphatase (ALP), serum glucose, serum urea nitrogen (BUN)

URINALYSIS: Yes
- Time schedule for collection of urine: urine was sampled at Week 7 and 13, respectively.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight (16 h prior to urine sampling)
- Parameters checked: pH, glucose, protein, occult blood, bilirubin, ketones, colour (visual), specific gravity

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals (adrenals, brain, oesophagus, eye, heart, hind leg muscles, kidneys, large intestine, liver, lung, ovaries, mesenteric lymph nodes, pancreas, sciatic nerve, skin, small intestine, spinal cord, spleen, sternum, stomach, testes, thyroid, trachea, urinary bladder, uterus and unusual lesions and tissue masses)
HISTOPATHOLOGY: Yes, all animals (adrenals, brain, oesophagus, heart, kidneys, liver, lung, pancreas, spleen, sternum, testes, trachea, uterus and unusual lesions and tissue masses)
Absolute and relative organ weights of adrenals, brain, heart, kidney, liver, lung, spleen, testes and uterus were determined in all animals.
Statistics:
The quantitative data derived from this study were subjected to statistical analysis by the Student’s t-test. Qualitative data from urinalysis were analysed using the Fisher’s Exact test. All statistical comparisons were made at the 95% confidence level (p = 0.05).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
all dose levels: unthrifty hair/coat (non-adverse)
Mortality:
mortality observed, treatment-related
Description (incidence):
all dose levels: unthrifty hair/coat (non-adverse)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
865 mg/kg bw/day (Week 7): stat. signif. increase in haematocrit and MCV in males (non-adverse); 3460 mg/kg bw/day (Week 13): stat. signif. increase in neutrophil/lymphocyte ratio in males (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
All dose levels: inconsistent stat. significant changes in glucose, ALP and BUN in males and/or females (non-adverse); for details see "Any other information on results incl tables"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
865 mg/kg bw/day (m): increase in protein (Weeks 7 and 13) and specific gravity (Week 7); 1730 mg/kg bw/day: decrease in pH (f; Week 7) and specific gravity (m; Week 13); 3460 mg/kg bw/day (m): increase in protein (Week 13); all non-adverse
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
3460 mg/kg bw/day: signif. increase in abs. and rel. heart and liver weight (f), increase in abs. liver weight (m) (non-adverse); 865 mg/kg bw/day: signif. decrease in abs. and rel. spleen weights (f), increase in abs. kidney weight (m) (non-adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
3460 mg/kg bw/day: scattered yellow nodular lesions of all lobes of lung in 1 male (non-adverse); 3640 and 1730 mg/kg bw/day (m, f): varying degrees of unthriftness of the hair of the hindquarters (non-adverse)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
3460 mg/kg bw/day (f): slight increase in frequency and severity of cytoplasmic vacuolation in liver (non-adverse); scattered yellow nodular lesions of all lobes of the lung in 1 male (non-adverse)
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities were observed during the entire study period. At 3460 mg/kg bw/day, animals exhibited unthrifty hair on their headquarters from Day 3 until termination of the study. In all cases, the hair appeared matted and oily. However, this did not appear to be irritating or distressful to the animals. The same symptoms were observed in animals treated with 1730 mg/kg bw/day during Week 6 of the study. The number of animals exhibiting unthrifty coats in this group varied on a weekly basis, and continued throughout the remainder of the study. Two male rats in the low dose group (865 mg/kg bw/day) also developed unthrifty coats during Weeks 9 and 10 of the study.

BODY WEIGHT AND WEIGHT GAIN
Weekly mean body weights and total weight gains for all treatment groups were comparable to those of controls throughout the study.

HAEMATOLOGY
At Week 7, a statistically significant increase in haematocrit and MCV was observed in males treated with 865 mg/kg bw/day. A statistically significant increase in neutrophil/lymphocyte ratio in males was noted at 3460 mg/kg bw/day (Week 13). These differences, while statistically significant compared to controls, were considered to be of no toxicological importance. The magnitude of these changes was within the normal range of values established in this strain of rats.

CLINICAL CHEMISTRY
In males and females treated with 3460 mg/kg bw/day, serum glucose values statistically significantly decreased at Week 7 of the study. The decrease in glucose at week 7 was also observed in females receiving the mid (1730 mg/kg bw/day) and low dose (865 mg/kg bw/day). In contrast, a statistically significant increase in glucose values at 865 and 1730 mg/kg bw/day was noted in males at Week 7 and 13, respectively. Although there were several statistically significant changes in glucose, these changes were not considered to be of toxicological significance, since they were neither unidirectional nor time- and dose-related. In addition, the serum values at Week 7 for both the control males and females were on the high side of the normally observed range, which enhanced the apparent difference between these values and depressed the glucose values at Week 7 in males and females receiving 3460 mg/kg bw/day. Furthermore, statistically significant increase in serum alkaline phosphatase (ALP) were observed in males treated with 3460 mg/kg bw/day (Weeks 7 and 13). In contrast, decreases in ALP were noted in females receiving 1730 mg/kg bw/day (Week 13) and 865 mg/kg bw/day (Week 13). It was concluded that the inconsistent changes in glucose and ALP were attributable to the increased lipid uptake resulting from the uptake of the substance, and thus of no toxicological relevance. The significant increase in serum urea nitrogen values in males treated with 865 mg/kg bw/day (Week 13) was within the normal historical range of this values in this strain of rats and without dose-response relationship, and thus not considered to be of toxicological relevance.

URINALYSIS
At 865 mg/kg bw/day, a statistically significant increase in protein (Weeks 7 and 13) and specific gravity (Week 7) was observed in males compared to controls. At 1730 mg/kg bw/day, a statistically significant decrease in pH (Week 7) in females and specific gravity (Week 13) in males was noted. At the highest dose level (3460 mg/kg bw/day), urinary protein was increased in males at Week 13 of the study. However, these changes were considered to be of not toxicological importance, since they occurred in the absence of any corresponding adverse effects.

ORGAN WEIGHTS
At 3460 mg/kg bw/day, a statistically significant increase in absolute and relative heart and liver weight in females was observed. The increase in liver weights of females was probably associated with the slight increase in the frequency and severity of hepatic cytoplasmic vacuolation observed in these animals. At the same dose level, absolute liver weights were also significantly increased in males. At 865 mg/kg bw/day, a statistically significant decrease in absolute and relative spleen weights in females and an increase in absolute kidney weight in males was noted. However, none of the weight changes in liver and kidney of males and spleen and heart of females was considered to be of toxicological relevance, since no significant histopathological findings were reported for these organs.

GROSS PATHOLOGY
At 3640 mg/kg bw/day, all males and females exhibited varying degrees of unthriftness of the hair of the hindquarters. Three males and two females from the mid dose group (1730 mg/kg bw/day) showed a similar condition. However, these effects were not considered to be of adverse nature. One male rat treated with 3640 mg/kg bw/day exhibited scattered yellow nodular lesions of all lobes of the lung associated with an aspirated foreign body type of pneumonia. There were no other gross changes attributable to treatment with the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
A slight increase in the frequency and severity of hepatic cytoplasmic vacuolation was observed in females treated with 3640 mg/kg bw/day, which was accompanied by the increased in absolute and relative liver weights, but was not considered pathological significant. One male rat treated with 3640 mg/kg bw/day exhibited scattered yellow nodular lesions of all lobes of the lung, indicative of an aspirated foreign body type of pneumonia. Only non-treatment-related microscopic changes were observed in the remaining tissues evaluated from the treated rats of all dose groups. The frequency and severity of these changes were similar between control and treated animals. Histopathological changes observed in the lungs and the upper respiratory tract of both control and treated rats were consistent with those seen in chronic murine pneumonia. Incidental microscopic changes were seen in other tissues of both control and treated rats (e.g. in kidney, spleen and pancreas).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 460 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOEL
Effect level:
3 460 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: histopathology (females): slight increase in the frequency and severity of hepatic cytoplasmic vacuolation accompanied by an increase in absolute and relative liver weights (non-adverse)
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate and reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common functional groups, common precursors/breakdown products and similarities in physicochemical and toxicological properties (refer to endpoint discussion for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
(EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for grouping of substances and read-across

There are no data available on the repeated dose toxicity of isodecyl pivalate (CAS 60209-82-7). In order to fulfil the standard information requirements set out in Annex IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of repeated dose toxicity

CAS

Chemical name

Molecular weight

Repeated dose toxicity oral

60209-82-7 (a)

Isodecyl pivalate

ca. 242

WoE:

RA: CAS 111-62-6
RA: CAS 58958-60-4

111-62-6 (b)

Ethyl oleate

310.51

Experimental result:
NOAEL ≥5500 mg/kg bw/day

58958-60-4

Isooctadecyl pivalate

354.61

Experimental result:
NOAEL ≥3460 mg/kg bw/day

(a) Substances subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in bold font.

(b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for isodecyl pivalate (CAS 60209-82-7). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

Subchronic oral toxicity

CAS 111-62-2

A 90-day oral feeding study was performed with ethyl oleate (CAS 111-62-6) according to the 1993 FDA draft "Redbook II" guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food) and presented in a publication (WoE, Bookstaff, 2004). The study protocol was similar to OECD Guideline 408. The purpose of the study was to determine the safety of ethyl oleate in a 91-day feeding study in Sprague-Dawley rats. 20 rats/sex/dose were administered ethyl oleate via the feed at levels of 0, 3.3, 6.7, and 10% by weight (approximately 0, 2000, 3900 and 6100 mg/kg bw/day in females, and 0, 1800, 3600 and 5500 mg/kg bw/day in males). All diets were calorie- and fat-matched, using high oleic safflower oil (HOSO) as the control fat. 1/20 low-dose males and 2/20 mid-dose died during the study, due to causes unrelated to the treatment. No treatment-related clinical signs were observed. Both the absolute body weight and body weight gain of the mid- and high dose group females were significantly lower (<10%) than the control group. However, rats on the ethyl oleate diets gained more weight during the course of the study than in historical control data. The lower body weight relative to control rats was directly related to lower food consumption relative to the controls. The food intake was lower due to lower palatability of the diet, but the change was not dose-related and is considered not to have toxicological relevance. 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. 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%). There were no treatment-related effects on haematology, clinical chemistry and urinary parameters. The ophthalmological examinations did not show treatment-related findings. The results of the neurological tests were comparable between the control group and treatment groups. No effects were noted on the spermatocytes in males and the estrous cycle in females. No gross pathological abnormalities were observed in the animals of the control and treatment groups. During the histopathological examination, 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.

The 90-day oral NOAEL was determined to be ethyl oleate as ≥ 10% of the dietary intake, which corresponds to approximately 5500 and 6100 mg/kg bw/day in males and females, respectively.

CAS 58958-60-4

The subchronic oral toxicity of isooctadecyl pivalate (CAS 58958-60-4) was investigated in a 13-week oral toxicity study in male and female rats, which was performed similar to OECD Guideline 408 (WoE, Avon, 1983). Groups of 10 animals per sex received the undiluted test substance daily (7 days/week) at dose volumes of 1.0, 2.0 and 4.0 mL/kg bw/day, corresponding to dose levels of 865, 1730 and 3460 mg/kg bw/day, respectively, based on a density of 0.865 mg/mL. A similar constituted group of animals was sham-gavaged and served as controls. No mortalities were observed during the entire study period. At all dose levels, animals of both genders exhibited unthrifty hair at varying times and degree throughout the study, which was not considered to be irritating of distressful to the animals, and thus of no toxicological concern. No changes in weekly determined mean body weights and the total body weight gain were observed in treated animals of any dose group compared to controls. Haematological analysis revealed an increase in haematocrit and MCV in males treated with 865 mg/kg bw/day and an increase in neutrophil/lymphocyte ratio in males at 3460 mg/kg bw/day. However, the magnitude of these changes was within the normal range of values established in this strain of rats, the statistically significant difference compared to controls was regarded to be of no toxicological importance. Several statistically significant changes in glucose values were observed in animals of all dose groups compared to controls at clinical chemistry analysis, but these changes were not considered to be toxicologically significant since they occurred neither unidirectional nor time- and dose-related. Furthermore, a statistically significant increase in serum alkaline phosphatase (ALP) was observed in males treated with 3460 mg/kg bw/day, whereas decreases in ALP were noted in females receiving 1730 mg/kg bw/day and 865 mg/kg bw/day, respectively. It was concluded that the inconsistent changes in glucose and ALP were attributable to the increased lipid uptake resulting from the uptake of the substance, and thus of no toxicological relevance. All other significant changes in clinical chemistry parameters (increase in BUN at 865 mg/kg bw/day) were within the normal historical range of this parameter in rats of this strain and without dose-response relationship, and thus not considered to be of toxicological relevance. The changes in urinary parameters (pH, protein and specific gravity) were not regarded to be of toxicological relevance, since they occurred in the absence of any corresponding adverse effects. Apart from treated animals showing varying degrees of unthriftness of the hair of the hindquarters, one male rat treated with 3640 mg/kg bw/day exhibited scattered yellow nodular lesions of all lobes of the lung associated with an aspirated foreign body type of pneumonia. There were no other gross changes attributable to treatment with the test substance. A statistically significant increase in absolute and relative liver weight in females treated with 3640 mg/kg bw/day was observed, which was probably associated with the slight, but not pathologically significant, increase in the frequency and severity of hepatic cytoplasmic vacuolation observed in these animals. None of the weight changes in liver and kidney of males and spleen and heart of females was considered to be of toxicological relevance, since no significant histopathological findings were reported for these organs. Apart from the histopathological changes in livers of female rats, only non-treatment-related microscopic changes were observed in the remaining tissues evaluated from the treated rats of all dose groups. The frequency and severity of these changes were similar between control and treated animals. Histopathological changes observed in the lungs and the upper respiratory tract of both control and treated rats were consistent with those seen in chronic murine pneumonia. Incidental microscopic changes were seen in other tissues of both control and treated rats (e.g. in kidney, spleen and pancreas). Since no adverse effects were observed up to and including the highest dose level administered, the NOAEL for male and female rats was considered to be ≥ 3460 mg/kg bw/day.

Conclusions for repeated dose oral toxicity

In the subchronic (90-day) repeated dose toxicity study that was performed with the structurally similar substance ethyl oleate (CAS 111-62-6) in rats, no treatment-related systemic effects were observed up to and including the highest dose level (WoE, Bookstaff, 2004). The NOAEL was set at ≥ 10% ethyl oleate as dietary intake, corresponding to approximately 5500 and 6100 mg/kg bw/day in males and females, respectively. A 90-subchronic (90-day) repeated dose toxicity study was performed with isooctadecyl pivalate (CAS 58958-60-4), resulting in no systemic toxicity in male and female rats up to and including the highest dose level of 3460 mg/kg bw/day (WoE, Avon, 1983).

Based on these results, the overall NOAEL for subchronic toxicity was considered to be ≥ 3460 mg/kg bw/day.

Justification for classification or non-classification

Based on read-across from the structurally similar substances, the available data on repeated dose toxicity via the oral route do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.