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Description of key information

In a 28-day repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, the No Observed Adverse Effect Level (NOAEL) of N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was considered to be 150 mg/kg bw/day in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June 2008 - 20 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline 407 without any deviation
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Germany
- Age at study initiation: 33 ± 1 day
- Weight at study initiation (mean): 161.6-165.3 g (male); 123.0-129.1 g (female)
- Housing: Housed together (5 animals per cage) in H-Temp (PSU) cages
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat “GLP”, meal (Source: Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): Drinking water (from water bottles), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10 air changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 10 June - 18 July 2008
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: A specified amount of the test substance was weighed and mixed with water using a magnetic stirrer. The frequency of the test substance preparations was weekly.

VEHICLE
- Concentration in vehicle: 0, 0.1, 0.5 and 1.5 g/100 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical method: Stability, homogeneity and verification of the concentrations of the test substance were analysed in separate studies (Study no.: 08L00155, 08L00185 and 08L00228) using Gas Chromatograph equipped with flame ionisation detector (FID).
- Results: Analysis results demonstrated the stability of the test substance preparations over a period of up to 7 days at room temperature. It showed the homogeneous distribution of the test article in drinking water and the correctness of the prepared concentrations (96-107 % of the nominal concentrations).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days/week
Remarks:
Doses / Concentrations:
0, 10, 50 and 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of the results of a 14-day preliminary study (Study No. 10S0589/07090) in which the same test item was administered to Wistar rats by gavage at dose-levels of 300 and 1000 mg/kg bw/day. Several severe clinical findings were observed even at 300 mg/kg bw/day including premature death. 150 mg/kg bw/day, chosen as highest dose, was the half of lethal dose.
- Rationale for animal assignment (if not random): Animals were grouped randomly using a computer.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- Mortality and morbidity: Twice daily on working days and once daily on Saturdays, Sundays and public holidays
- Clinical signs: Once daily before and after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Once before group allocation, on the first day of treatment and then once a week until the end of the study and before sacrifice.

FOOD CONSUMPTION:
- Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

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
- Individual drinking water consumption was determined weekly over a period of 4 days and calculated as mean water consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of the administration period the eyes of all animals and on Day 28 the eyes of the control (0 mg/kg bw/day) and high dose animals (150 mg/kg bw/day) were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic (Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retro-orbital venous plexus from fasted animals at the end of the treatment period.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- Parameters checked (haematology): Leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), Differential blood count, reticulocytes and prothrombin time
- Parameters checked (clinical chemistry): Sodium, potassium, chloride, calcium, inorganic phosphate, urea, creatinine, glucose, total bilirubin, total proteins, albumin, globulin, cholesterol, triglycerides, magnesium, alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), γ-glutamyltransferase (GGT) and alanine aminotransferase (ALAT)

URINALYSIS: Yes
- Time schedule: Urine was collected overnight and urinalysis was performed in all animals towards the end of the treatment period.
- Metabolism cages used for collection of urine: Yes, animals were individually placed in metabolism cages
- Animals fasted: Yes, overnight
- Parameters checked: Color, volume, turbidity, sediment examination, specific gravity, pH, protein, glucose, ketones, bilirubin, blood and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: A functional observational battery (FOB) was performed in all animals at the end of the administration period. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests.
Battery of functions tested:
- Home cage observations: Posture, tremor, convulsions, abnormal movements, impairment of gait and other findings
- Open field observations: Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes and number of rearings within two minutes
- Sensorimotor Tests/Reflexes: Approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other findings
- Motor activity of each animal was also measured using TSE Labmaster System over a 1 h period.
Sacrifice and pathology:
- GROSS PATHOLOGY: On completion of the treatment period, after at least 16 h fasting, all surviving animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
- ORGAN WEIGHTS: Body weight of each animal was recorded before sacrifice and the following organs were weighed: liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus and thyroid glands.
- HISTOPATHOLOGY: For all animals, the following tissues were preserved in neutral-buffered 4 % formaldehyde solution: brain, pituitary gland, thyroid glands, parathyroid glands, thymus, esophagus, salivary glands (mandibular gland, sublingual gland), trachea, lungs, pharynx, larynx, nose (nasal cavity), aorta, heart, liver, pancreas, spleen, kidneys, adrenal glands, testes, ovaries with oviducts, uterus, vagina, epididymides, prostate, seminal vesicle, stomach (fore- and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric and axillary lymph node), sciatic nerve, bone marrow (femur), eyes, extraorbital lacrimal glands, skin, female mammary gland, spinal cord (cervical, thoracic and lumbar cord), sternum with marrow, femur with knee joint and skeletal muscle.
After fixation the following tissues of control and high-dose groups were processed for microscopic examination: brain, pituitary gland, thyroid glands, parathyroid glands, thymus, esophagus, salivary glands (mandibular gland, sublingual gland), trachea, lungs, aorta, heart, liver, pancreas, spleen, kidneys, adrenal glands, testes, ovaries with oviducts, uterus, vagina, epididymides, prostate, seminal vesicle, stomach (fore- and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric and axillary lymph node), sciatic nerve, bone marrow (femur), eyes, skin, female mammary gland and spinal cord (cervical, thoracic and lumbar cord).
Other examinations:
None
Statistics:
- Body weight and body weight change: A comparison of each group with the control group was performed using Dunnett's test (two-sided) for the hypothesis of equal means.
- Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, footsplay test, and motor activity; clinical pathology parameters, urine volume, and urine specific gravity; weight parameters in pathology: Non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value ≤ 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Urinalysis, except color, turbidity, volume and specific gravity: Pairwise comparison of each dose group with the control group using Fisher's exact test for the hypothesis of equal proportions.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, 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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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 premature deaths occurred during the study.
- At 150 mg/kg bw/day, salivation was observed in all male and female animals on few days between Day 11 and 28, and respiration sounds were observed after treatment in 3 male animals and 4 female animals on several days between Day 7 and 28. These findings were assessed as adverse and related to the test article in the study report. However, it is considered by expert judgement that these sporadic and transient effects are not considered as adverse and probably due to an unpleasant taste of the substance as often observed with chemical compounds. This hypothesis is also considered by the authors of the report of the OECD 421 study (Reproduction/Developmental toxicity screening test) in which same findings are observed without increased incidence or frequency at dose-levels up to 200 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN:
- Body weight was significantly increased (+10.6 %) in male animals at 10 mg/kg bw/day on Day 28, only. This single occurrence only observed in low dose males was without any correlative to food consumption or other clinical parameters measured. Therefore, this finding was assessed as incidental and not related to treatment with the test substance.

FOOD AND WATER CONSUMPTION:
- No treatment-related changes were noted.

FOOD EFFICIENCY:
- No treatment-related changes were noted.

OPHTHALMOSCOPIC EXAMINATION:
- No treatment-related changes were noted.

HAEMATOLOGY:
- No significant treatment-related changes were noted.

CLINICAL CHEMISTRY:
- No significant treatment-related changes were noted.

URINALYSIS:
- No significant treatment-related changes were noted.

NEUROBEHAVIOUR:
- No treatment-related changes were noted.

ORGAN WEIGHTS:
- Absolute weights: When compared to control group, the females of the 10 mg/kg body weight group revealed significantly changed kidney weights. In the higher dose groups no significant deviations from the control group were observed. Therefore, this is regarded to be an incidental finding and not related to treatment. All other mean absolute weight parameters did not show significant differences when compared to the control groups.
- Relative weights: When compared to control group, the mean absolute weights of brain were statistical significantly reduced (9, 7 and 8 % reduction at 10, 50 and 150 mg/kg bw/day, respectively). The relative brain weight was changed in all treated females. No dose response relationship and no histopathologic correlate were observed. Therefore it is regarded to be a spontaneous finding and not related to treatment. All other mean relative weight parameters did not show significant differences when compared to the control groups.

GROSS PATHOLOGY AND HISTOPATHOLOGY:
- All gross lesions occurred singly or they were biologically equally distributed between control and treatment groups.
- All findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them are considered to be incidental or spontaneous in origin and without any relation to treatment.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: as stated in the study report
Critical effects observed:
not specified

None

Conclusions:
Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was considered to be 150 mg/kg bw/day in the male and female Wistar rats.
Executive summary:

In a 28-day repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was administered daily to groups of Wistar rats (5/sex/dose) at doses of 0 (vehicle), 10, 50 and 150 mg/kg bw/day over a period of 28 days by gavage. Examinations during the study included: mortality, clinical signs, functional observation battery (including motor activity), body weight change, food and water consumption, ophthalmological examinations, hematology, blood chemistry, urinalysis, organ weights, macroscopic examination and histopathology.

No deaths occurred during the study and only slight signs of toxicity were observed at 150 mg/kg bw/day: salivation was observed in all male and female animals on few days between Day 11 and 28, and respiration sounds were observed after treatment in 3 male animals and 4 female animals on several days between Day 7 and 28. No significant treatment-related changes were noted in the body weight gain, food and water consumption, ophthalmological examinations, hematological parameters, urinalysis and neurotoxicological parameters at any dose-level. No treatment-related effects were noted on organ weights or necropsy and microscopic findings.

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was considered to be 150 mg/kg bw/day in the male and female Wistar rats by expert judgement (stated 50 mg/kg bw/day in the study report) .

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1

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

In a 28-day repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was administered daily to groups of Wistar rats (5/sex/dose) at doses of 0 (vehicle), 10, 50 and 150 mg/kg bw/day over a period of 28 days by gavage. Examinations during the study included: mortality, clinical signs, functional observation battery (including motor activity), body weight change, food and water consumption, ophthalmological examinations, hematology, blood chemistry, urinalysis, organ weights, macroscopic examination and histopathology.

No deaths occurred during the study and only slight signs of toxicity were observed at 150 mg/kg bw/day: salivation was observed in all male and female animals on few days between Day 11 and 28, and respiration sounds were observed after treatment in 3 male animals and 4 female animals on several days between Day 7 and 28. These findings were assessed as adverse and related to the test article in the study report. However, it is considered by expert judgement that these sporadic and transient effects are not adverse and probably due to an unpleasant taste of the substance as often observed with chemical compounds. This hypothesis is also considered by the authors of the report of the OECD 421 study (Reproduction/Developmental toxicity screening test) in which same findings are observed without increased incidence or frequency at dose-levels up to 200 mg/kg/day. No significant treatment-related changes were noted in the body weight gain, food and water consumption, ophthalmological examinations, hematological parameters, urinalysis and neurotoxicological parameters at any dose-level. No treatment-related effects were noted on organ weights or necropsy and microscopic findings.

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was considered to be 150 mg/kg bw/day in the male and female Wistar rats by expert judgement (stated 50 mg/kg bw/day in the study report).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Well performed study according to OECD 407 guideline and under GLP protocol.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No repeated dose toxicity study via inhalation is available. N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) is a liquid with a vapour pressure around 1.78 10-7 Pa at 25°C (EPI suite estimation). Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur, and no subacute inhalation test is needed.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Exposure via inhalation is unlikely.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The substance is corrosive for skin. Therefore, dermal route is not the preferred route for repeated dose toxicity studies.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The substance is corrosive for skin. Therefore, dermal route is not the preferred route for repeated dose toxicity studies.

Justification for classification or non-classification

Based on the available results (Kaspers 2009) and according to the criteria laid down in EU regulation (EC) n° 1272/2008/EC (CLP) and EU directive67/548/EEC (DSD), the substance is not classified for repeated dose toxicity.