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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.03.2013 - 15.08.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
other: CD / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: males 44 days, females 45 days (at 1st day of test item administration)
- Weight at study initiation: males 199.2 to 236.0 g, females 153.8 to 196.3 g (at 1st day of test item administration)
- Housing: individually, Makrolon cages type III plus
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 16 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
Administration volume: 4 mL/kg bw/day
The amount of the test item to be administered was adjusted daily to each animal's most recently recorded body weight up to the end of test week 6 and once weekly thereafter.
The control animals received the vehicle at the same administration volume daily in the same way.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tests were conducted to determine the concentration and homogeneity of the test item in the formulations using a high performance liquid chromatography (HPLC) method with subsequent UV detection. The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity and stability.
The content of TAC in the Test Item formulations was quantified applying a calibration curve calculated from defined peak areas of TAC.
Duration of treatment / exposure:
Treatment: 90 days
Post treatment (recovery): 56 days
Frequency of treatment:
Once daily for 90 days
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 30 and 120 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
100 animals (50 males and 50 females)
Main study animals:
80 (40 male and 40 female) animals, 10 animals per sex and group in groups 1 to 4
Recovery period animals:
20 (10 male and 10 female) animals, 5 animals per sex and group in groups 1 and 4
Additionally, 10 reserve animals (5 males and 5 females) were available for possible replacement during the adaptation period.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels for this study had been selected by the Sponsor based on the results of a 28-day dose-range-finding study in rats (LPT study
no. 28634).
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Clinical signs:
Animals were observed individually at least once daily for any signs of behavioural changes, reaction to treatment, or illness. In addition, the animals were checked regularly throughout the day by cage-side observations. The onset, intensity and duration of any signs observed were recorded individually for each animal.
Additionally, detailed clinical observations were made for all animals outside the home cage in a standard arena. The observations were recorded before the first administration (on test day 1, to allow for within-subject comparisons) and once weekly throughout the treatment period (always on day 7 per test week for test weeks 1 to 12 and on day 6 of test week 13).

Mortality:
Animals were checked early in the morning and again in the afternoon to look for dead or moribund animals.

Body weight:
The body weight of each rat was recorded at the time of group allocation (predose), on the day of first administration (test day 1) and once a week thereafter, always on the same day of the week throughout the experimental period.

Food and drinking water consumption:
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each animal in each group on completion of a treatment week.
The drinking water consumption of each animal was monitored daily by visual appraisal throughout the study.

Neurological screening:
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad), as well as assessment of grip strength (Meyer) and motor activity was conducted for all main study and recovery animals.
Observational screening: 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 manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.
Functional screening: grip strength, locomotor activity.

Haematology:
Haemoglobin content, erythrocytes, leucocytes, haematocrit value, reticulocytes (relative), platelets, differential blood count (relative and abolute), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), thromboplastin time
(TPT), activated partial thromboplastin time (aPTT).

Clinal chemistry:
Albumin, globulin, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (in blood), calcium, chloride, potassium, sodium, alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT).

Urinalysis:
Volume, pH, colour, turbidity, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite.
Microscopic examination: epithelial cells, leucocytes, erythrocytes, organisms, further costituents (i.e. sperm, casts), crystalluria.

Ophthalmological examination:
An ophthalmological examinations were performed on all main study and recovery animals prior to the first administration, at the end of the treatment period (main study animals), and at the end of the recovery period (recovery animals), each time before the blood sampling for laboratory examinations was performed.
The following ocular structures were examined: Adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus.
Sacrifice and pathology:
The main study animals were dissected following a randomisation scheme on test day 91 or test day 92. Main study animals not dissected on test day 91 were dosed again on that day. Necropsy of all animals allocated to the recovery period was performed on test day 147. The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically .

Gross necropsy:
All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
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. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs was recorded.
The weights of the following organs of all animals were determined before fixation: adrenal gland, brain, epididymis, ovary, testicle, heart, kidney, liver, spleen, thymus.
Blood smears were prepared from all animals scheduled for haematology examinations.

Histopathology:
The following organs were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining:
Adrenal gland, aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), caecum, epididymis, eye with optic nerve and harderian gland, gall bladder, heart (left and right ventricle, septum), injection site (i.v. injection), small intestine (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), large intestine (colon, rectum), kidney and ureter, liver, lungs (with mainstem bronchi and bronchioles), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary, pancreas, pituitary, prostate and seminal vesicles with coagulating glands, salivary glands (mandibular, parotid, sublingual), seminal vesicle, skin (left flank), spinal cord (3 sections), spleen, stomach, testicle, thymus, thyroid (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), tongue (incl. base), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts), vagina.
If parathyroids could be identified macroscopically, they were examined microscopically if in the plane of section and in cases they were noted as grossly enlarged. The livers of all animals of group 2 (low dose) and group 3 (intermediate dose) were examined histologically.
A detailed histomorphological examination was performed on one testicle and one epididymis of all male animals of group 1 and group 4 (with special emphasis on the qualitative stages of the spermatogenesis and the histopathology of the interstitial testicular structure).
All observations upon final assessment were reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings were recorded, reported and archived.
Other examinations:
Sperm viability and morphology (spermiogram):
One epididymis and one testicle were used for the sperm count. The sperm viability was determined and the sperm morphology was examined for all male animals (n = 50) according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Statistics:
Students t-test: all numerical functional tests / sperm count

Multiple t-test based on DUNNETT, C. W.: Body weight / Food consumption / Haematology / Clinical biochemistry / Relative and absolute organ weights

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
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:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality:
No premature deaths were noted at oral treatment with 10, 30 or 120 mg TAC/kg bw/day.

Clinical signs:
120 mg TAC/kg b.w./day: all males and females revealed pilo-erection, reduced motility, ptosis and salivation. Pilo-erection was observed during the entire treatment period. Reduced motility, ptosis and salivation were noted on various test days. Reduced motility and ptosis started within 10 minutes after administration and lasted up to 24 hours. Salivation occurred within 5 minutes after administration lasting up to 5 minutes.
Additionally, prone position was noted occasionally for a few animals. Further, all male and female animals revealed burrowing of their noses in the bedding material immediately after administration from test day 5 onwards. All clinical signs previously observed had completely subsided.
10 or 30 mg TAC/kg bw/day: no effects.

Food and drinking water consumption:
120 mg TAC/kg bw/day: relative food consumption was transiently reduced by 18% for the males and by 23% for the females in test week 1 compared to the control group. No test item-related influence was noted on the food intake at the high dose from test week 2 onwards.
10 or 30 mg TAC/kg bw/day: no effects.
The visual appraisal of the drinking water consumption did not reveal any test item-related changes for the treated animals in comparison to the control animals, neither during the main study nor during the recovery period.

Clinical biochemistry:
120 mg TAC/kg bw/day: serum level of cholesterol was increased by 35% in the females. The male high-dose animals were not affected. After the recovery period the serum level of cholesterol appeared to be still increased by 21% in the previously high-dosed females.
10 or 30 mg TAC/kg bw/day: no effects.

Neurological screening:
120 mg TAC/kg bw/day: a few test item-related changes in comparison to the control animals during observational screening within 2 hours after dosing. The changes observed included a slight waddling gait, a lack of normal response to toe or tail pinch, an impaired ability for wire maneuver, increased positional passivity, and a lowered resistance on limb rotation. The spontaneous motility of the high-dosed animals was reduced by 30% for the males and by 39% for the females. After the recovery period no differences were noted for the parameters of the observational screening and the functional tests between the previously high-dosed animals and the control animals.
10 or 30 mg TAC/kg bw/day: no effects.

Organ weights:
120 mg TAC/kg bw/day: relative liver weight of the males and the relative and the absolute liver weight of the females was increased by up to 29%. After the recovery period the relative and the absolute liver weight of the females was still increased by 12% and by 16%, respectively. The relative and the absolute liver weight of the males was at the level of the control group.
10 or 30 mg TAC/kg bw/day: no effects.

Sperm viability and morphology: no effects at any dose group.

Histopathology (restricted to the high-dose group and the liver of the low and intermediate dose groups):
Microscopic evaluation of the high-dosed animals revealed a test item-related change in the liver of the males (hepatocellular hypertrophy, centrilobular/midzonal). This (centrilobular) hepatocellular hypertrophy was statistically significantly different compared to the male controls and was not observed in the female high dose animals. After the recovery period one of five males of the high dose recovery showed a fatty change in centrilobular and midzonal locations in the liver on test day 147 which was confirmed by moderate Oil red O-staining. This is considered to be a late-onset effect after end of treatment.
Histopathological evaluation of the liver of the males and females of the low and intermediate dose groups did not reveal any test item-related changes.
No test item-related effects were noted in the testes and the epididymides of the male animals and in the female reproductive organs at the high dose.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Dose descriptor:
LOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral treatment with TAC for 90 days did not cause any effects of biological or toxicological significance up to a dose level of 30 mg TAC/kg bw/day.
An oral dose of 120 mg TAC/kg bw/day for 90 days led to pilo-erection, reduced motility, ptosis, salivation and/or prone position, reduced body weight, effects on functional observational battery parameters, increased serum cholesterol levels, increased liver weights, and morphological lesions in the liver in form of a centrilobular hypertrophy in male and/or female animals. However, the vast majority of changes noted had completely subsided at the end of the 8-week recovery period.
No effects were noted on the reproductive organs of male and female animals.
The no-observed-adverse-effect level (NOAEL) was considered to be 30 mg TAC/kg bw/day following 90 days of oral exposure.