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Administrative data

Description of key information

NOAEL (OECD 408, oral, rat): ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar Crl: Wi/Br
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 ± 1.5
- Humidity (%): 65 ± 10
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- 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:
yes
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
Remarks:
Doses / Concentrations:
100, 500, 1000 mg/kg bw/day (corresponding to 1200, 6000, 12000 ppm in the diet)
Basis:
nominal in diet
No. of animals per sex per dose:
10
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:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Individually in weekly intervals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- 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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Individually in weekly intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- 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.

HAEMATOLOGY: Yes
- 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.

CLINICAL CHEMISTRY: Yes
- 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.

URINALYSIS: Yes
- 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:
ORGAN WEIGHTS:
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.
Statistics:
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
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
not treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
not treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
not treatment-related
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
not treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related effects were observed. 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 GAIN
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)
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
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
Water consumption was unaffected by treatment. No significant intergroup differences were noticed compared to the controls.

OPHTHALMOSCOPIC EXAMINATION
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.

HAEMATOLOGY
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 CHEMISTRY
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
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.

ORGAN WEIGHTS
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 PATHOLOGY
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.

HISTOPATHOLOGY: NON-NEOPLASTIC
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.

HISTORICAL CONTROL DATA (if applicable)
Historical control data for hematology, clinical chemistry and urinalysis were provided.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All endpoints
Key result
Critical effects observed:
not specified

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) was determined as NOEL. 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (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

Oral

Subacute

There is one study available on the subacute oral toxicity of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) in female Holtzman rats via oral gavage, which was not performed according to a specific guideline (Levenstein, 1962). During 5 consecutive days, 10 animals were daily administered the test substance in olive oil in 8 dose intervals of 1 mL total volume, amounting for a daily total dose of approx. 5000 mg/kg bw/day. During the treatment and a subsequent 23-day post-exposure recovery period, no mortalities and clinical signs of toxicity were noted. The food consumption and body weight gain appeared normal in treated animals during the entire study period. At sacrifice, no gross pathological and histopathological abnormalities were noted in any of the treated animals. Based on the results of this study, the NOEL of the test substance in female rats was considered to be ca. 5000 mg/kg bw/day.

 

Subchronic

The subchronic oral toxicity of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) was investigated in male and female Wistar Crl: Wi/Br rats in a GLP-conform study according to OECD 408 (Korn, 1988). Based on a preliminary 4-week dose-range finding test, the test substance was administered continuously for a period of 13 weeks to groups of 10 animals per sex and group at dietary concentrations of 1200, 6000, 12000 ppm, corresponding to reported doses of 100, 500 and 1000 mg/kg bw/day in males and females, respectively. A similar constituted control group received the plain diet. During the study period, 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. However, since this spontaneous finding has been observed in rats of this strain before, and no pulmonary changes were noted in any other animal of this study, the observed mortality was considered to be coincidental and not attributable to treatment. No clinical signs of toxicity were observed in any of the other animals during the 13-week study period. Body weights as well as food and water consumption in treated animals were not significantly altered compared to those of controls. At ophthalmoscopic examination after 6 weeks and at study termination, no adverse findings on eyes attributable to treatment were noted. Haematological and clinical chemistry analysis revealed sporadic differences in some parameters which were not dose-related and thus considered to be incidental and not toxicologically relevant. All mean values of haematological and clinical chemistry parameters were in the range of normal values compared to historical controls. Urinalysis and determination of organ weights showed no treatment-related difference between treated and control animals. Gross necropsy and histopathological examination, including male and female reproduction organs, did not reveal any findings indicative of treatment-related effects. Some incidental spontaneous changes in gross pathology and histopathology were nearly equally distributed among control and dose group animals, and thus of no toxicological relevance. Based on the overall effects of the study, a NOAEL of ≥ 1000 mg/kg bw/day for male and female rats was derived.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study is the most adequate and reliable study with the longest duration and the lowest dose descriptor.

Justification for classification or non-classification

The available data on repeated dose toxicity of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) via the oral route do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC and are therefore conclusive but not sufficient for classification.