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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(1998)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Stability in vehicle: confirmed in analytical testings conducted under GLP (confirmed for at least 8 days)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU
- Source: Harlan Laboratories BV, Kreuzelweg 53, 5961 Horst, The Netherlands
- Age at study initiation: 6-7 weeks
- Weight at study initiation (mean): males 168.3 g (131-189 g), females 148.4 g (132-174 g)
- Housing: in groups of 2 or 3 animals per cage in Makrolon cages Type IV
- Diet and water: ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): approx. 55
- Air changes (per hr): >/= 10
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The administration volume was 2 ml/kg body weight.

Before the start of treatment, the suitability of the proposed formulation was confirmed by the analyses of concentration and stability of dosage forms. The formulation was prepared as needed and taking into account the analytically determined stability. Analyses for homogeneity were not necessary as the formulations were solutions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and stability of the test item in the administration vehicle were checked prior to study start. The concentration of samples of control and each test substance dosage form prepared were determined tree times during the study using capillary gas chromatography. Quantification was done by internal standard calibration. Analyses during the study verified that the test item content agreed with the target concentrations within the defined limits.
Duration of treatment / exposure:
93 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200 and 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a 4-week study conducted according to OECD Guideline 407 (Report No. 28785, 1999; also included in chapter repeated dose toxicity).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
General condition and behavior of all animals were checked and recorded daily about 1 hour after administration.
Inspections on mortality and morbidity of all animals were performed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes, all animals
A careful clinical examination including observation outside the home cage in a standard arena was performed once prior to treatment and in a weekly interval up to necropsies in all animals. Any clinical signs and abnormalities were recorded.

BODY WEIGHT: Yes, all animals
The individual body weights for all animals were determined just before first treatment of animals and daily thereafter. Furthermore, body weights were recorded immediately before scheduled necropsies, for calculation of relative organ weights.

FOOD CONSUMPTION AND WATER CONSUMPTION: Yes, all animals
Food and water consumption per cage were determined weekly. These data were then used to calculate the group means for each period of approximately 7 days. The weight of the food offered at the start of the measurement period minus the weight of the food at the end of the period is defined as the food consumption of the animal in g.
Comparable calculations were done for the water intake.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before start of treatment (all animals); at the end of treatment (control and high dose group)

HAEMATOLOGY: Yes, all animals
- Time schedule for collection of blood: Week 13
The blood samples were collected in the morning from the retro-bulbar venous plexus of non-fasted animals anesthetized with CO2/O2 (80/20).
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), thrombocytes, Hepato Quick, differential blood count.

CLINICAL CHEMISTRY: Yes, all animals
- Time schedule for collection of blood: Week 13
The blood samples for determination of glucose concentrations were taken from the caudal vein of non-fasted, non-anesthetized animals.
The blood samples used for determination of the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasted animals anesthetized with CO2/O2 (80/20).
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, albumin, protein (total), cholesterol, creatinine, urea, glucose, sodium, potassium.

URINALYSIS: Yes, all animals
- Time schedule for collection of urine: Week 13
Urine samples were collected at room temperature during a period of 16 hours. During the urine collection periods, water was offered ad libitum but feed was not supplied.
- Parameters checked: Quantitatively: Volume, Density, Crea, Prot, Crea x Vol, Prot x Vol; Semiquantitatively: pH, glucose, blood, bilirubin, ketone bodies, urobilinogen; Microscopy of sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
- Time schedule: Week 14
All animals scheduled for necropsies and all moribund animals were sacrificed by exsanguination under isoflurane anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination.
At study termination the following organs of animals sacrificed scheduled were weighed before fixation: Adrenals, Brain (3 regions), Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Uterus with Cervix.

HISTOPATHOLOGY: Yes
The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution and were histopathologically evaluated for control and all treated groups:
Abnormalities, Kidneys, Thyroid/ Parathyroids.

The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution and were histopathologically evaluated for control and high dose group:
Adrenals, Aorta, Brain (cerebrum, cerebellum, brain stem), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femur (with bone marrow/joint), Haderian glands, Heart, Ileum, Jejunum, Larynx, Liver, Lungs, Lymph nodes (mandibular, mesenteric, popliteal), Ovaries/Oviducts, Pancreas, Peyer's patches, Pituitary gland, Prostate, Rectum, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulating glands), Skin (mammary region), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow, Stomach, Testes, Thymus, Tongue, Trachea, Uterus with Cervix, Urinary bladder, Vagina.

The following organs, tissues or representative pieces of them were fixed in 10 % neutral buffered formalin or Davidson's solution:
Exorbital lacrimal glands, Eyelids, Gall bladder, Head with skull cap, Nasal cavities, Optic nerves, Pharynx, Skeletal muscle, Tongue, Urethra, Ureters, Vagina, Zymbal glands, Physical identifier.
Statistics:
The statistical evaluation of data related to clinical chemistry, hematology, body and organ weights will be performed using routines of the validated Pristima System.
Statistical evaluations on body weight and organ weight data will be done using the Dunnett-test.
Clinical chemistry and hematology data will be evaluated using a Dunnett test, an adjusted U-test or an adjusted Welsh test.
All variables that are not dichotomous are described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
Statistical tests will not be performed for groups, which are smaller than 3.
For statistical evaluations of histopathological findings, if any, the PATHDATA program was used.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
Survival was not affected by the treatment with the test substance. At clinical observations some findings were observed (decreased motility, increased salivation, resistance during administration, high stepping gait, erect tail, tense, agitated, vocalization), but were not regarded to be of toxicological relevance as these findings were observed only for single animals and/or without dose dependence and/or only for a short period of time.

BODY WEIGHT AND WEIGHT GAIN
Body weight development of males and females was not retarded by the treatment up to 1000 mg/kg.

FOOD CONSUMPTION AND WATER CONSUMPTION
Food intake in treated groups was comparable to that in the respective control group.
Water intake of males treated with 1000 mg/kg was slightly increased. Water intake in the other groups was roughly comparable to that of the respective control group. The differences to control were slight and no corresponding findings were observed. Therefore, a toxicological relevance is not inferred from these data.

OPHTHALMOSCOPIC EXAMINATION
No evidence for treatment-related effects.

HAEMATOLOGY
Hematology gave no evidenced for treatment-related effect on red or white blood or blood coagulation.
The only conspicuous findings were significantly increased HQuick time at 1000 mg/kg in males and decreased number of basophils in this dose group. However, as these differences were slight, HQuick time in females was comparable to the control group and the basophil count in females was slightly changed in the opposite direction and no further obviously corresponding findings were observed, these slight changes are considered not to be of toxicological relevance.

CLINICAL CHEMISTRY
Determination of enzymes, substrates and electrolytes in peripheral blood gave no evidence for toxicological relevant effects. The only conspicuous finding was a statistically significant increased activity of aspartate aminotransferase in males treated with 1000 mg/kg. However, as the difference to control was slight and no correlating findings were observed (e.g. changes in liver morphology) this change was not regarded to be of toxicological relevance.

URINALYSIS
Quantitative urinalysis revealed no treatment-related effects. The increase in creatinine concentration in females at 1000 mg/kg is not considered to be of toxicological relevance as the excreted amount of creatinine was comparable to that of the control group.
With the exception that there was a very slight tendency for increase of ketone bodies and urobilinogen at 1000 mg/kg in the dipstick test, the semi-quantitatively determined blood, bilirubin, glucose and protein concentrations as well as microscopy of sediment were unremarkable in all groups.

ORGAN WEIGHTS
Absolute and relative weight of kidneys were slightly (13.6%) but statistically significantly increased in females treated with 1000 mg/kg. Furthermore, absolute and relative weights of uterus were decreased starting at 200 mg/kg. As there were no clear dose dependence and the histopathology gave no evidence for treatment-related effects a toxicological relevance was not assumed.
Further differences of the mean organ weights compared to the respective control are also not regarded to be of toxicological relevance as the differences were slight and no obvious correlating findings were observed, e.g. at histopathology.

GROSS PATHOLOGY
At necropsy no treatment-related findings were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related histopathological findings were noted in the kidneys of animals treated with 1000 mg/kg.
In the kidneys, cortical tubular vacuolation (proximal and distal convoluted tubules) was present in 7/10 males and in 8/10 females at 1000 mg/kg.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Kidney findings (slight increased absolute and relative weight, cortical tubular vacuolation) at 1000 mg/kg

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The investigation gave evidence for treatment-related effects on kidneys. Histopathology showed cortical tubular vacuolation (at proximal and convoluted renal tubules) at 1000 mg/kg. The observed renal cortical tubular vacuolation was not accompanied by any further degenerative components, and is considered to be due to a storage process. The very slight tendency for increase of ketone bodies and urobilinogen at 1000 mg/kg observed at urinalysis and the slightly but statistically significantly increased absolute and relative kidney weights in females might correlate with these findings.

Applicant's summary and conclusion

Executive summary:

A subchronic repeated dose toxicity study was conducted according to OECD TG 408. The test substance was administered orally by gavage to 10 male and 10 female Wistar rats per dose group in daily doses of 0, 40, 200, and 1000 mg/kg bw for a period of 3 months.

Survival was not affected by the treatment with the test substance. At clinical observations no relevant findings were observed. Body weight development as well as mean food and water intake in treated groups did not point to a toxicologically relevant effect.

Histopathology showed in the kidneys cortical tubular vacuolation (at proximal and convoluted renal tubules) at 1000 mg/kg, which is considered to be due to a storage process. The very slight tendency for increase of ketone bodies and urobilinogen at 1000 mg/kg and the slightly but statistically significantly increased absolute and relative kidney weights in females might correlate with these findings.

Neither hematology, clinical chemistry, observations at necropsy, determination of organ weights nor histopathological evaluation of organs gave evidence for further treatment-related effects up to 1000 mg/kg.

Thus, under the conditions described the NOAEL is 200 mg/kg body weight for the substance.