Propanamide, N,N',N''-1,3,5-benzenetriyltris[2,2-dimethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study conducted in compliance with GLP regulations; reliable without restriction

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Details on strain: Crl:WI(Han)
- Source: Charles River Labs, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: means: 159.8 - 161.5 g (males); 129.2 - 132.2 g (females)
- Housing: In groups of five in Tecniplast H-Temp polysulfonate cages, with bedding and enrichment
- Diet: Ground Kliba mouse/rat maintenance diet ad libitum, supplier: Provimi, Kaiseraugst, Switzerland
- Water: Community Local tap-water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
The food in the food hoppers was changed weekly. The test-substance preparations were mixed every 4 weeks.
- Mixing appropriate amounts with (Type of food):
For each concentration, the test substance was weighted out and mixed with a small amount of food (ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland). Then corresponding amounts of food, depending on test group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the food matrix was demonstrated for 32 days. The test item was found to be homogeneously distributed. Concentration measurements showed a maximum range of 97.9 - 107.6% of nominal concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously (ad libitum in the diet)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1500, 5000, 15000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Males: 0, 90.9, 305.8, and 960.9 mg/kg bw/day; females: 0, 115.1, 361.1, and 1104.4 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Dose selection based upon previously conducted 28-day study in rats

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality twice daily, once daily during weekends and holidays; clinical observations daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to start of treatment and weekly thereafter.
In an off-cage arena, the following parameters were checked: abnormal behaviour in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, pupil size, assessment of feces and urine discharged during the examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to and on the first day of treatment, weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to and towards the end of treatment
- Dose groups that were examined: control and high dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: towards the end of treatment
- Anaesthetic used for blood collection: Yes: isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Parameters checked: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time (Hepato Quick’s test; HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: towards the end of treatment
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), magnesium (MG)

URINALYSIS: Yes
- Time schedule for collection of urine: towards the end of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Parameters checked: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, colour, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observational battery (FOB) towards the end of the treatment period, consisting of home cage and open field observations, sensory motor tests/reflexes, motor activity
- Dose groups that were examined: all animals
- Battery of functions tested:
- Home cage observations: posture, tremors, convulsions, abnormal movements, gait abnormalities
- Open field observations: behaviour on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes / pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements / stereotypes, gait abnormalities, activity / arousal level, faeces excreted within 2 minutes (number / appearance / consistency), urine excreted within 2 minutes (amount / colour), rearing within 2 minutes
- Sensory motor tests / reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behaviour during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
- Motor activity assessment: The examinations were performed using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany). For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals for 5 minutes in each case. For each rat, measurement was started individually when the 1st beam was interrupted and finished exactly 1 hour later.

Sacrifice and pathology:

- Sacrifice by decapitation under isoflurane anesthesia followed by gross pathology.
- Organ weighed: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.
- Histopathology from control and high-dose animals from the following organs: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Esophagus, Eyes with optic nerve, mammary gland, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes) Ovaries, Pancreas, Parathyroid glands, Peyer’s patches, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Statistics:

- Body weight: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- FOB, Clinical Pathology, Organ weights: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study.
No test substance-related clinical findings were observed.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes of body weight and body weight change were observed in any test group.
The significantly lower body weight change value on study day 21 (-11%) in male animals of test group 3 (15000 ppm) was assessed as being incidental and not related to treatment as no significant differences were observed on other study days.

FOOD CONSUMPTION AND COMPOUND INTAKE
No test substance-related effects on food consumption were obtained. Over the entire application period deviations in food consumption values observed for male and female animals of test groups 1-3 (1500, 5000 and 15000 ppm) were assessed as being incidental and not related to treatment.

WATER CONSUMPTION
No test substance-related findings were observed.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related findings.
All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.
In females of test group 1 (1500 ppm) relative monocyte counts were higher compared to controls, but this parameter was not dose-dependently changed and, therefore, this alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
In males of test group 1 (1500 ppm) triglyceride values were higher compared to controls, but this parameter was not dose-dependently changed and, therefore, this alteration was regarded incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.
In males of test group 1 (1500 ppm) higher counts of transitional epithelial cells were found in the urine sediment. In females of the same test group, the urinary pH value was higher compared to controls. Both parameters were not dose-dependently changed and, therefore, these alterations were regarded as incidental and not treatment-related.

NEUROBEHAVIOUR
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose response relationship or occurred in single rats only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:
- Home cage observations: No findings, which could have been assessed as being related to treatment and of toxicological concern, were observed.
- Open field observations: No findings, which could have been assessed as being related to treatment and of toxicological concern, were observed.
- Sensorimotor tests/reflexes: No findings, which could have been assessed as being related to treatment and of toxicological concern, were observed.
- Motor activity measurement: Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female rats.

ORGAN WEIGHTS
- Spleen: When compared with the control group 0 (set to 100%), the mean absolute spleen weight in male animals of test group 3 (15000 ppm) was significantly (*) decreased (1500 ppm: 92%, 5000 ppm: 101%, 15000 ppm: 91%*). The change in spleen weight was considered incidental as there was no clear dose-response relationship, there were no statistically significant changes in relative spleen weights and there were no histopathological findings in the spleen.
All other mean absolute weight parameters did not show significant differences when compared to the control group.

None of the mean relative weight parameters showed significant differences when compared to the control group 0.

GROSS PATHOLOGY
Two female animals of test group 2 (5000 ppm) showed dilation of uterine horns which was confirmed on histopathological examination and represents a normal physiological state during the rat estrus cycle, pathologically relevant findings were not noted in this organ. All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to the highest dose of nominal 15000 ppm (equivalent to 961 mg/kg bw/day for males and 1104 mg/kg bw/day for females).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

DISCUSSION

The test substance was administered for 3 months daily via the diet to male and female Wistar rats at concentrations of 0, 1500, 5000 and 15000 ppm (test groups 0-3).

With regard to clinical examinations, no signs of general systemic toxicity in Wistar rats of either sex were observed.

Concerning clinical pathology, no treatment-related, adverse findings were observed up to a concentration of 15000 ppm.

Regarding pathology, no treatment-related findings were noted in Wistar rats of either sex.

 

CONCLUSION

In conclusion, the oral administration of the test substance via the diet over a period of 3 months revealed no signs of toxicity in male and female Wistar rats up to a concentration of 15000 ppm.

Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 15000 ppm for male (961 mg/kg bw/d) and female Wistar rats (1104 mg/kg bw/d).

Applicant's summary and conclusion

Conclusions:

In conclusion, the oral administration of Propanamide, N,N',N''-1,3,5-benzenetriyltris[2,2-dimethyl- (9CI)] via the diet over a period of 3 months revealed no signs of toxicity in male and female Wistar rats up to a concentration of 15000 ppm. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 15000 ppm for male (961 mg/kg bw/d) and female Wistar rats (1104 mg/kg bw/d).