N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Oct 2005 - 24 May 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:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted September 21, 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

EC Number:

262-679-1

EC Name:

N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Cas Number:

61260-55-7

Molecular formula:

C24H50N4

IUPAC Name:

N1,N6-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Age at study initiation: males: 26 days, females: 27 days
- Weight at study initiation: males: 68 - 81.3 g, females: 68.4 - 81.8 g
- Housing: individually in MAKROLON cages type III
- Diet (ad libitum): commercial Ssniff R/M-H V1530-1534
- Water (ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item-vehicle mixture was freshly prepared every day. The test item was dissolved in the vehicle to the appropriate concentrations and was administered orally by gavage at a constant volume daily for 90 days. The control animals received the vehicle for 90 days in the same manner. The amount of the test item was adjusted to each animal´s current body weight daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance-vehicle mixture analysis was conducted at study initiation and at study termination. The samples were analysed according to a gas chromatographic method.

Duration of treatment / exposure:

90 test days plus 6 additional test weeks for the animals scheduled for the recovery period.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
44, 110, 275 mg/kg bw/day
Basis:
other: nominal conc.

No. of animals per sex per dose:

10 in main group
5 in recovery group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the 28-day dose-range-finding study in rats (LPT study no. 19518/05). In this dose-range-finding study dose levels of 40, 120 and 360 mg test substance/kg bw/d were tested. 40 mg/kg bw/d was regarded to be the NOAEL, starting at 120 mg/kg bw/d clinical signs of toxicity were noted in form of pilo-erection. At 360 mg/kg bw/d further signs of toxicity were noted in form of a reduced body weight in males and hematological and biochemical changes in both sexes.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: signs of behavioral changes, reaction to treatment or illness, skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first exposure and once a week thereafter (1, 2, 4, 8 and 24 hour after administration)
Signs noted included changes in skin, fur, eyes, mucous membranes, occurence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of group allocation, on the day of commencement of treatment and once a week thereafter, always on the same day of the week throughout the experimental period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examination of all animals prior to the start of administration, at main study termination and at the end of the recovery period.
- Parameters checked: adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination and at the end of the recovery period
blood samples were taken from the retrobulbar venous plexus under light ether anesthesia from animals fasted overnight
- How many animals: main study: all animals; recovery: all animals
- Parameters checked: HGB, RBC, WBC, differential blood count (absolute and relative), Reti, PCT, HCT, TPT, aPTT, MCV, MCH, MCHC

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination and at the end of recovery
Blood samples were taken from the retrobulbar venous plexus under light ether anesthesia from animals fasted overnight.
- How many animals: all main study animals and all animals scheduled for the recovery period.
- Parameters checked: albumin, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), blood urea nitrogen, calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: at main study termination and at the end of the recovery period
Urine samples were collected from animals fasted overnight. The urine was collected for 16 hours in an URIMAX funnel cage.
- Parameters checked: pH, specific gravity, volume, protein, glucose, bilirubin, urobilinogen, ketones, hemoglobin, nitrite, epithelial cells, leucocytes, erythrocytes, organisms, further constituents, crystalluria

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In test week 12/13 approx. 1-2 hours after dosing and before any blood sampling for laboratory examinations, screening of sensory reactivity to stimuli of different types, as well as the assessment of grip strength and motor activity assessment were conducted in all main study animals.
- Battery of functions tested: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function, grip strength, locomotor activity,

OTHER:

Oestrus cycle: At the end of the main study and at the end of the recovery period, vaginal lavages were taken daily from all female animals for periods 2 weeks each. The stage of estrus cycle observed in each vaginal lavage was recorded.

Sperm count and spermatogenic staging:
Staging of spermatogenesis was conducted by histological examination of one testicle and epididymis of all male animals of groups 1-4. From the other testicle and epididymis a sperm count was carried out during necropsy. The sperm viability was determined and the sperm morphology was examined for the control and for the treated animals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: all superficial tissues were examined visually and by palpation. The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
HISTOPATHOLOGY: Yes: adrenal gland, aorta abdominalis, bone marrow, brain, epididymis, eye with optic nerve, gross lesions, heart, intestine, kidney and ureter, liver, lungs, lymph nodes, mammary gland, nerve, oesophagus, ovary, pancreas, pituitary, prostate, salivary glands, skin, spinal cord, spleen, stomach, testicle, thymus, thyroid, tissue masses or tumors, trachea, urinary bladder, uterus, vagina

Statistics:

The test substance groups were compared to the control group. The following statistical methods were used: Student's t-test for analysis of data on all numerical functional tests, urinalysis, haematology (recovery), clinical biochemistry (recovery), oestrus cycle and sperm parameter (differences considered statistically significant if p ≤ 0.01); multiple t-test based on Dunnett for analysis of data on body weight, food consumption, haematology, clinical biochemistry, absolute and relative organ weights (differences considered statistically significant if p ≤ 0.01), Exact test of Fisher for analysis of data on histology and sperm parameter (differences considered statistically significant if p ≤ 0.05).

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
44 mg/kg bw/d: no effects
110 mg/kg bw/d: 1 male and 1 female revealed a laboured breathing on one or two test days in test week 10, 11 or 8, respectively. Further, pilo-erection was noted for the same female animal on the same test day of test week 8.
275 mg/kg bw/d: reduced motility for male and female animals, laboured breathing and pilo-erection were noted in nearly all animals on several test days,salivation for two animals (male and female), the same animal revealed an abdominal position on four test days.
recovery: all changes noted for animals (275 mg/kg/bw/d) had subsided immediately at the beginning of the recovery period.
275 mg/kg bw/d: 3 of 15 male animals died prematurely during the day on test days 21, 26, 52. additionally, 3 of 15 females died on test days 16 or 22. Four of these animals revealed a laboured breathing starting one or two days before their premature deaths. All other animals survived.

BODY WEIGHT AND WEIGHT GAIN
44 and 110 mg/kg bw/d: no effects (male and female)
275 mg/kg/bw/d: reduced body weight in males from test week 1 onwards up to 13% compared to the control. Body weight gain was reduced accordingly.

FOOD CONSUMPTION
44 mg/kg bw/d: no effects (male and female)
110 or 275 mg/kg/bw/d: reduced food consumption in females from test week 1 onwards by up to 16 or 20% in test week 10 or in test weeks 2, 5, 6, 7, 8 and 11, respectively.
recovery: changes observed for the females had subsided by the end of the 6-week recovery period.

HAEMATOLOGY
44 and 110 mg/kg bw/d: no effects (male and female)
275 mg/kg: leucocytes (WBC) +69%, neutrophilic granulocytes (abs.) +106%, lymphocytes (abs.) +90%, large unstained cells (LUC) (abs.) +103% in females
recovery: no test substance-related changes noted for the previously high dosed animals (male and female)

CLINICAL CHEMISTRY
44 and 110 mg/kg bw/d: no effects (male and female)
275 mg/kg bw/d: slightly increased activity of alkaline phosphatase in the male animals (+31%) on test day 91 compared to control
recovery: changes noted for the male animals with 275 mg/kg bw/d had subsided at the end of the recovery period

GROSS PATHOLOGY
Oral treatment with 44, 110, 275 mg/kg bw/d caused ulcerous lesions in the stomach in several animals. The effect appeared to be dose-related but only in male animals. Further, an increased lobular pattern was noted in the liver of one male and one female treated with 110 mg/kg bw/d and in three animals treated with 275 mg/kg bw/d.

HISTOPATHOLOGY: NON-NEOPLASTIC (male and female)
Histomorphological examination revealed an ulcerative gastritis in the fore-stomach and purulent tracheitis in male and female rats treated with 110 or 275 mg/kg bw/d and a purulent laryngitis in the animals treated with 275 mg/kg bw/d. These local changes are considered due to the corrosive properties of the test item. No systemic histopathological changes were noted.
At the end of the 6-week recovery the forestomach showed a mild recovery and the trachea and the larynx a complete reversibility of the lesions observed. Spermatogenic examination (staging) did not reveal any test substance-related influence.

Evaluation of the estrus cycle and sperm count/viability/morphology: No test substance-related influence was noted.

No effects occurred for neurobehavior, urinalysis, water consumption, ophthalmoscopic examination.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

> 275 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

NOAEL

Effect level:

44 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results of the analysis showed that the test substance-vehicle mixtures were correctly prepared and the concentration, stability and homogeneity found were in good agreement to those expected. The results of 92.2% to 107.5% of the nominal value were well within the admissible limits.

Applicant's summary and conclusion

Conclusions:

All changes observed were considered to be due to the local corrosive changes noted, causing behavioural, biochemical and haematological changes and in some cases mortality. The changes observed were not considered to be due to systemic toxicity of the test substance given by gavage but due to its corrosive properties. Under consideration of all changes noted and the corrosive properties of the test substance, the NOAEL for systemic changes was above 275 mg/kg bw/day; the NOAEL for local changes was 44 mg/kg bw/day.