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Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
Limit test:

Test material

Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 1
Chemical structure
Reference substance name:
Molecular formula:
Constituent 2
Reference substance name:
oleyl palmitamide
oleyl palmitamide

Test animals

other: Wistar Crl: Wi/Br
Details on test animals or test system and environmental conditions:
- Source: Versuchstierzucht Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: about 5 weeks
- Weight at study initiation: males 129-170 g, females 115-138 g
- Fasting period before study: not applicable
- Housing: Individually in Makrolon cages; bedding "Altromin Laboreinstreu", Altromin GmbH, Lage, Germany: produced from soft pure wood, dried, disdusted and sterilized at 180 °C, renewed weekly.
- Diet: Ssniff R pelleted diet (standard laboratory rat diet, Ssniff Spezialdiaeten GmbH, Soest, Germany), ad libitum.
- Water: Tap water from Makrolon drinking bottles, ad libitum.
- Acclimation period: 7 days

- Temperature (°C): 21.5 ± 1.5
- Humidity (%): 65 ± 10
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
Details on oral exposure:
- Rate of preparation of diet (frequency):
Feed/test compound mixtures were prepared for the first 6 study weeks and again for the 6-13 weeks study phase. Fresh batches were provided at the same time for all test groups at the beginning of treatment and again 6 weeks later.

- Mixing appropriate amounts with (Type of food):
A sample of the test substance was submitted to the producer of the standard laboratory diet, where it was incorporated into the basal diet Ssniff R10. Therefore, appropriate amounts of test compound were weighed out for each concentration level and mixed with small amounts of the basal diet (300 g). Due to the granular structure of the test compound, these first admixtures were pulverised in a grinder. Mixing was continued in three steps to the final quantity. Each preparation was then pelleted, packed into paper bags, labelled with the study project-no., the diet number, compound concentration, testgroup, and production date and forwarded to IBR.

- Storage temperature of food: Ambient
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentrations and homogeneity of the test substance in the diet were determined by both IR spectroscopy and by gas chromatography separately for the dose range finding study, the first part of the feeding study and the second part of the feeding study.

The test substance contents in the diets for part 1 and 2 of the study were found to be within 10% of the target contents, and homogeneity was within 10% of the average of samples. In the test compound/rat diet mixtures for the preliminary dose range finding study the average test compound contents were within 15% of the target contents.
Stability during storage at ambient temperatures was tested over periods of 6 weeks, 3 months and 4 months; the results demonstrate that the average test compound content did not deviate more than 7% of the originally determinated average content and prove the stability of the test substance in rat diet admixtures at room temperature storage.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously in the diet
Doses / concentrationsopen allclose all
Dose / conc.:
1 200 ppm
corresponding to daily dose level of 100 mg/kg bw/day
Dose / conc.:
6 000 ppm
corresponding to daily dose level of 500 mg/kg bw/day
Dose / conc.:
12 000 ppm
corresponding to daily dose level of 1000 mg/kg bw/day
No. of animals per sex per dose:
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The chosen test compound concentrations were determined on the basis of results obtained from a preceding 4-week dose range finding study performed with N-Oleyl Palmitamide levels of 1000, 5000, 10000 and 50000 ppm. Results of this dose range finding study indicated very likely possible nutritional effects at a 5% dietary concentration (50000 ppm), for instance increased gains in weight and significantly increased food consumption. It was therefore decided to use a 1.2% dietary concentration (high dose) in the 13-weeks study.

Positive control:


Observations and examinations performed and frequency:
- Time schedule: daily, viability or mortality twice daily, dose responses recorded weekly
- Cage side observations included: sensory and motor behaviour, hair coat, body orifices, urine and fecal excretion, general health status, dose responses

DETAILED CLINICAL OBSERVATIONS: Yes, hearing (by simple noise production) and reflex-examinations (modified IRVING-Screen) with special regard to awareness, emotion, coordination, and autonomic functions.
- Time schedule: Prior to initiation, after 6 weeks and at termination.

- Time schedule for examinations: Individually in weekly intervals

- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/animal/week: Yes
- Daily compound intake calculated from the group mean food consumption and mean body weight: Yes

- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

- Time schedule for examinations: Individually in weekly intervals.

- Time schedule for examinations: Prior to initiation, after 6 weeks and at termination.
- Dose groups that were examined: In 10 males and 10 females of each group.

- Time schedule for collection of blood: After 6 weeks and at termination.
- Anaesthetic used for blood collection: No data, blood was taken from retrobulbar plexus.
- Animals fasted: No data
- How many animals: 10 males and 10 females of each test group.
- Parameters examined: Erythrocytes (RBC), leukocytes (WBC), hemoglobin, hematocrit, platelet count, MCV, MCH, MCHC, differential blood count, reticulocytes, inclusion bodies, thrombocytes, prothrombin time.

- Time schedule for collection of blood: After 6 weeks and at termination.
- Animals fasted: No data
- How many animals: 10 males and 10 females of each test group.
- Parameters examined: Albumin, alk. phosphatase, total bilirubin, calcium, chloride, cholesterol, urea nitrogen (BUN), creatinine, glucose, AST (GOT), ALT (GPT), inorg. phosphorus, iron, potassium, sodium, total protein, triglyceride, uric acid, GOT/GPT-ratio and Na/K-ratio (by statistical evaluation). Electrophoresis: albumin, alpha1 + alpha2-globulin, beta-globulin, gamma-globulin.

- Time schedule for collection of urine: After 6 weeks and at termination.
- Metabolism cages used for collection of urine: Yes, animals housed in metabolism cages for 18 hours after water administration.
- Animals fasted: No data, intragastric administration of 20 mL/kg of water.
- Parameters examined: Colour, protein, pH-value, glucose, bilirubin, urobilinogen, blood, nitrite, ketones, sediment, specific gravity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; animals in moribund condition or mortalities and survivors of the study. Animals were sacrificed by CO2 asphyxiation and exsanguinated. A complete autopsy was performed for each animal, an examination of the cranial, thoracic, abdominal and pelvic cavity was carried out, abnormal findings were recorded.
HISTOPATHOLOGY: Yes; all dissected organs were preserved in buffered 10% formaldehyde solution except of kidneys, pancreas, testes with epididymides and an additional sample of the liver, which were fixed in Bouin's solution. Eyes were fixed in Zenker's solution. Samples of all tissues determined for histopathological examination were trimmed, embedded in tissue wax and stained with hematoxylin and eosin. From heart, liver, and adrenals additionally frozen sections were prepared and stained with Sudan III for fat content judgements.
Samples from all tissues listed below were removed from all animals, blocks and slices were prepared from the control and the high dose group and examined histopathologically: skin, mammary gland, salivary gland, trachea, esophagus, thyroid (2x), parathyroid (2x), thymus, heart, lung, aorta, pituitary, tongue, liver, spleen, pancreas, kidney (2x), adrenal (2x), stomach, duodenum, jejunum, ileum, cecum, colon, seminal vesicle, lymph node (mesenteric + cervical), ovary (2x)/testis + epididymis (2x), prostate/uterus + vagina, urinary bladder, sciatic nerve, skeletal muscle, bone with marrow (sternum + femur), eye with N. opticus (2x), cerebrum with brain stem, cerebellum, spinal cord (2x), lacrimal gland (2x), macroscopic changes.
Other examinations:
From all males and all females of all groups: Cerebrum with cerebellum, pituitary, heart, liver, kidneys (l + r), adrenal (l + r), spleen, prostate gland, testis (l + r) with epididymis, ovary (l + r), uterus, seminal vesicle.
Statistical analyses of data were performed separately for male and female animals. For the evaluation of weight changes, food consumption and water consumption a one- respective two-factorial analysis of variance was performed. To compare the group mean values the method of "Scheffé" was employed. The organ weights were evaluated by analysis of co-variance. Hereby the animal weight is the independent variable, the organ weight the dependent one. The comparison of the mean values was performed by the method of "Scheffé" for the analysis of co-variance.
Values of clinical chemistry and hematology were analysed as follows:
a) Analysis of variance for dose-effect curves with the factors group and time and the interaction group/time. The degrees of freedom for the factor time and the interaction group/time were corrected according to Greenhouse and Geisser (Epsilon-correction).
b) Mean values were compared according to the method of Scheffé after a preceding analysis of co-variance. The comparison (of the mean values) was carried out by correction with analysis of co-variance in such a manner as if the curves originated from the same starting-point.
c) If there were available one point time values only, an analysis of variance with subsequent Scheffé test for analysis of variance was performed.
The following significance levels were calculated:
p<0.05 slightly significant
p<0.01 significant
p<0.001 highly significant

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal signs were observed in any animal.
mortality observed, non-treatment-related
Description (incidence):
One male treated with 1000 mg/kg bw/day died after 13 weeks shortly prior to necropsy due to a severe pulmonary edema and hydrothorax. This spontaneous condition had occurred rarely in untreated control animals in this laboratory. The mortality was considered to be coincidental and not attributable to treatment, since there was no indication of such pulmonary changes in any other animal in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant intergroup differences in bodyweight gains during the 13-week study period. Slight variations among groups were within a range of ±10% to the controls and there was no evidence of dose-relation. The test substance was not considered to influence body weight development in any dose group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was comparable among the groups. There were no significant differences to the controls or dose-related group trends attributable to test compound intake. The actual intake of the test compound was within ±11% of the intended target dose for each group.
Food efficiency:
no effects observed
Description (incidence and severity):
The food conversion ratio was near to equal between control and dose group. No influence of the test compound was noticed throughout the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was unaffected by treatment. No significant intergroup differences were noticed compared to the controls.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes in eye structures with respect to cornea, sclera, lens and retina were observed. No significant changes were detected between control and dose group animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related hematology changes were noticed at either sex after 6 or 13 weeks.
Some sporadic differences were not dose-related and thus considered to be incidental. All mean values were in the range of normal, compared to historical controls. This refers also to total leukocyte-values, which were significantly increased in female groups after 13 weeks. There was, however, no dose-related relationship and a low control mean value. Thus the significant differences in total leukocytes are not considered to be treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
All mean values were found to be within normal ranges, compared to historical controls. Occasionally occurring significant differences to the concurrent control group were not specifically dose-related and therefore incidental findings, i.e. independent of treatment. The significant changes in triglycerides (females) were within normal ranges and not considered to be of biological importance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related differences between control and dose groups. The significant decrease of specific gravity in the high-dose group males after 6 weeks was not dose-related and no similar change occurred after 13 weeks.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were comparable between control and dose groups. There were no statistically significant intergroup differences or any dose-related group trends attributable to the test compound.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any findings indicative for treatment-related effects. Some incidental spontaneous changes were nearly equally distributed among control and dose group animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic evaluation of representative organ sections from all control and high-dose animals did not reveal any test compound-related changes. Some spontaneous incidental lesions or lesions due to sacrifice were found evenly distributed among control and high-dose animals of both sexes.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Key result
Critical effects observed:

Any other information on results incl. tables


Historical control data for hematology, clinical chemistry and urinalysis were provided.

Applicant's summary and conclusion

The test substance had no adverse effect on any of the tested parameters in rats of the Wistar-strain over the entire test period of 13 weeks.

Thus a dietary concentration of 12000 ppm (corresponding to a nominal dose of 1000 mg/kg bw/day) revealed a clear "no-effect" level. Significant intergroup differences in individual parameters were generally within normal limits and without any dose-relationship. These differences were therefore considered to be without biological importance and not attributable to test substance exposure.