Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06.03.2006 - 04.05.2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(July 1995)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-Ethylpiperidin
- Physical state: Colorless fluid
- Analytical purity: 100 %
- Lot/batch No.: 0102562158
- Expiration date of the lot/batch: January 17,2008
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Wistar (Hsd Cpb:WU)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan GmbH, Borchen, Germany
- Age at study initiation: About 6 weeks
- Housing: 3 or 2 per Makrolon cage Type IV
- Diet: Provimi Kliba 3883.0.15 pellets supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Demineralized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation of Formulation(s): Twice weekly at room temperature
Formulation(s) Stable for a Period of: 4 Days, the stability (0.5 mg/mL and 30 mg/mL concentration) of the test item in the vehicle was determined and confirmed by analytical examinations before the start of the study. The formulation was a clear solution. Therefore, no tests on homogeneity were done. The test item contents (all concentrations including vehicle control formulation) were checked at begin and near termination of the study.

Administration Volume: 10 mL/kg bw

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 20, 80 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected according to results obtained in a oral 2-week rat gavage study, were each three males and three females were treated with 0, 10, 50, 100 or 200 mg/kg bw/day N-Ethylpiperidin in water.
Thereby, 200 mg/kg bw was shown to be highly toxic indicated by several clinical symptoms of poor general condition (males) or reduced motility (both sexes), body weight stagnation (males) or depressed body weight development (females), which was caused by the adverse effects of the test substance on the fore stomach (keratolysis, epithelial hyperplasia, oedematous inflammation and ulceration), which were seen macro- and microscopically. As consequence of these symptoms all high dose rats were killed in moribund condition after 2 (males) or 11 (females) administrations as spontaneous death was expected.
Also in all rats of the 100 mg/kg bw dose group comparable adverse effects on the forestomach were evident, however, with lower degree. Mortality, clinical appearance, body weight development, and food/water intake and organ weights were not affected remarkably up to 100 mg/kg bw.
The number of leucocytes (males) and reticulocytes (females) was reduced from 50 mg/kg bw onwards.
The plasma alkaline phosphatase activity was decreased at 100 mg/kg bw in both sexes.
Therefore, the doses 5, 20, and 80 mg/kg bw were used in the present study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: for morbidity and mortality: Twice daily, once daily on weekends and public holidays; general clinical examinations (in cage): Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the start of treatment and once weekly thereafter, including observations in a standard arena (open field) for behavioral observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE: Weekly, per group
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, the blood samples were collected from the retro-orbital venous plexus
- Time schedule for collection of blood: Day 29, in the morning
- Anaesthetic used for blood collection: Yes, CO2/O2.
- Animals fasted: No
- How many animals: all animals per group
- Parameters examined:
• Leukocytes
• Erythrocytes
• Haemoglobin
• Haematocrit
• Reticulocytes
• Differential blood count
• Mean corpuscular volume (MCV)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• HQUICK
• Platelets

CLINICAL CHEMISTRY: Yes
- The blood samples for determination of glucose concentrations were taken in the morning on non-anesthetized animals from the caudal vein.
- The blood samples used for determining the other parameters in peripheral blood were also collected in the morning from the retro-orbital venous plexus of animals anesthetised with CO2/O2.
- Time schedule for collection of blood: Day 29
- Animals fasted: No
- How many animals: all animals per group
- Parameters examined:
• Alkaline phosphatase
• Aspartate aminotransferase
• Alanine aminotranferase
• Cholesterol
• Urea
• Creatinine
• Glucose
• Total protein
• Albumin
• Sodium
• Potassium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational Battery: Day 20-21 (relative), Motor activity: Day 23-24
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
• Functional Observation Battery (FOB)
Functional observations were performed near to the study termination. FOB observations were done earliest about 30 minutes after administration.
The following observations/examinations were performed:
- home cage observation:
posture, piloerection, gait abnormalities, involuntary motor movements, vocalization, others.
- observations during handling:
ease of removing reaction to being handled, muscle tone, palpebral closure, lacrimation, nasal discharge, salivation, stains and others.
- open field observations:
piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behaviour, gait abnormalities, vocalization, arousal, rearing, defecation, urination, others.
- The following manipulative tests were additionally performed:
approach response, touch response, auditory response, tail pinch response, pupil size, pupil response, righting reflex, grip strength and body temperature as well as landing foot splays.
• Motor Activity
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA).
- Motor activity was measured as the number of beam interruptions that occurred during the test session.
- Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal moved in the maze and interrupted one of the other beams.
- Habituation was evaluated as a decrement in activity during the test session.
- Determination of MA/LMA was done earliest about 30 minutes after administration in order of a random list (animal assignment list).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals alive at study termination and all rats to be sacrificed in moribund condition were killed by exsanguination under deep diethyl ether anesthesia and necropsied. Organs and tissues were subjected to thorough gross pathological examination and tissues were fixed.
Organ Weights
The weights of the brain, adrenals, heart, liver, spleen, thymus, kidneys, testes, and epididymides were recorded during the terminal necropsy. The organ weights were specified in both absolute and relative terms. The relative organ weights were calculated by normalization to 100 g body weight determined immediately prior necropsy of the pertinent animal.

HISTOPATHOLOGY: Yes,
The fixed material of rats was retained. Fixation, histological procedures as well as the number of organs and tissues fixed in 10 % neutral buffered formalin or Davidson's solution respectively.
Adrenal glands
Aorta
Brain (cerebrum, cerebellum, brain stem)
Epididymides
Esophagus
Eyes
Eyelids
Exorbital lacrimal glands
Femur (with joint)
Harderian glands
Head (with skull cap),
- Nasal Cavity
Heart
Intestine
- Peyer's patches
- Duodenum
- Jejunum
- Ileum
- Cecum
- Colon
- Rectum
Kidneys
Larynx
Liver
Lungs
Lymph nodes, mandibular
Lymph nodes, mesenteric
Lymph nodes, popliteal
Optic nerves (Brain parts fixed in formalin, eye parts fixed in Davidson’s solution
Ovaries
Oviducts
Pancreas
Pharynx
Pituitary gland
Prostate
Salivary glands (parotid, submandibular, sublingual)
Sciatic nerve
Seminal vesicles (incl. coagulating glands)
Skeletal muscle (thigh)
Skin (mammary region)
Spinal cord (cervical, thoracic, lumbar)
Spleen
Sternum with Bone Marrow
Stomach
Testes
Thymus
Thyroid glands (with parathyroids)
Tongue
Trachea
Ureters
Urethra
Urinary bladder
Uterus (with cervix)
Vagina
Zymballs glands
















Statistics:
- Body and organ weight data as well as food and water intake: Dunnett test in connection with a variance analysis.
- Biochemical parameters: analysis of variance followed by a Dunnett test, an adjusted Welch test or a Kruskal-Wallis test.
- All variables that were not dichotomous were described by sex, dose group and time point using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
- For the statistical evaluation of samples drawn from continuously distributed random variables three types of statistical tests were used, the choice of the test being a function of prior knowledge obtained in former studies.
- Provided that the variables in question were approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared more likely, a p value adjusted Welch test was applied. If the evidence based on experience with historical data indicated that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate.
- With respect to data collected in the functional observational battery categorical variables were analyzed with a repeated measures analysis of variance followed by a one-way analysis of variance. As part of the one-way analysis, contrasts were performed to compare the results of each treated group with those of the control group. The logic of the analysis plan for continuous variables was analogous to that of the categorical variables.
- In these types of statistical processing of measurement values a large number of comparisons are made, which may also lead to false-positive statements. On account of this problem for the evaluation not only the statistical significance but also the biological and toxicological relevance is considered.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- During the study no animal died in the study groups up to 80 mg/kg bw.
- No clinical findings were observed at cage side or detailed weekly clinical observations (including open field observations) up to 80 mg/kg bw.

BODY WEIGHT AND WEIGHT GAIN
- No statistically significant or dose-related changes in body weights or body weight gain were evident up to 80 mg/kg bw in males and females.

FOOD CONSUMPTION AND COMPOUND INTAKE
- No toxicologically relevant changes in food intake were observed up to 80 mg/kg bw in males and females

OPHTHALMOSCOPIC EXAMINATION
- No data

HAEMATOLOGY
- No toxicologically relevant changes in the red blood parmeters or in blood coagulation up to 80 mg/kg bw. Means among these parameters marked as statistically significant are of no toxicological relevance as dose dependences are missing and the deviations to control values are minimal.
There were also no statistically significant changes in white blood parameters up to 80 mg/kg bw.

CLINICAL CHEMISTRY
- No toxicologically relevant changes occurred in ASAT, ALAT and alkaline phosphatase activity in the plasma up to 80 mg/kg in both sexes:
ASAT (aspartate aminotransferase, males, day 29: 0 mg/kg bw: 83.7 U/l; 5 mg/kg bw: 89.4 U/l; 20 mg/kg bw: 74.7 U/l and 80 mg/kg bw: 75.3 U/l; females, day 29: 0 mg/kg bw: 82.5 U/l; 5 mg/kg bw: 75.3 U/l; 20 mg/kg bw: 77.7 U/l and 80 mg/kg bw: 68.9 U/l),
ALAT (Alanine aminotransferase, males, day 29: 0 mg/kg bw: 76.4 U/l; 5 mg/kg bw: 70.2 U/l; 20 mg/kg bw: 77.3 U/l and 80 mg/kg bw: 75.4 U/l; females, day 29: 0 mg/kg bw: 66.9 U/l; 5 mg/kg bw: 68.5 U/l; 20 mg/kg bw: 59.3 U/l and 80 mg/kg bw: 61.0 U/l), and
alkaline phosphatase activity in the plasma males, day 29: 0 mg/kg bw: 263 U/l; 5 mg/kg bw: 262 U/l; 20 mg/kg bw: 256 U/l and 80 mg/kg bw: 249 U/l; females, day 29: 0 mg/kg bw: 182 U/l; 5 mg/kg bw: 178 U/l; 20 mg/kg bw: 183 U/l and 80 mg/kg bw: 173 U/l) up to 80 mg/kg in both sexes.
- The levels of the plasma substrates were not toxicologically relevantly changed up to 80 mg/kg bw.
- The slightly lower (p ≤ 0.5) means for the creatinine concentration (20 and 80 mg/kg bw males (54 and 53 mcmol/L) and 80 mg/kg bw females (52 mcmol/L)) do not reflect a test substance-induced effect as all individual values are covered by the 2s-range (twice the standard deviation) of historical controls. In control males individual values were above the historical control range.
- The serum concentrations of sodium and potassium were not affected by the treatment up to 80 mg/kg bw. All individual values lay within the 2s-range of historical controls.

URINALYSIS
- No data

NEUROBEHAVIOUR
- The functional observation revealed no statistically significant different means for these measurements up to 20 mg/kg bw. For 80 mg/kg bw females, the mean landing footsplay and body temperature were statistically significantly reduced. Because these deviations were small and limited to one sex, they are considered to be incidental and without toxicological relevance.
- Mean and individual motor (MA) and locomotor (LMA) activity data for both the entire session (60-minute) MA and LMA and also the 10-minute intervals gave no indication of test-substance-related changes in motor or locomotor activity due to the treatment up to 80 mg/kg bw.

ORGAN WEIGHTS
- None of the absolute and relative organ weights were statistically significantly and/or dose-dependently changed up to 80 mg/kg bw.

GROSS PATHOLOGY
- No macroscopical organ changes attributable to the test substance were detected up to 80 mg/kg bw.

HISTOPATHOLOGY: NON-NEOPLASTIC
- There was one 80 mg/kg bw male exhibiting slight inflammation with keratolysis in the forestomach. This finding is most probably caused by a local irritation of the test substance as rats receiving 100 mg/kg bw in the two week pilot study revealed the same finding.
There was no evidence of any dose-related finding up to and including 20 mg/kg bw. From the viewpoint of pathology, the no-adverse-effect level is 20 mg/kg bw.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: local irritation (slight inflammation with keratolysis) observed in 1 of 5 males.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
> 80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse systemic effects were observed in the highest dose tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion