Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Description: Off-white solid
- Test substance storage: At room temperature in the dark
- Expiry date: 19 January 1999

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation:
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors.
- Diet: Standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least five days

DETAILS OF FOOD AND WATER QUALITY: Food: Each batch is analysed for nutrients, and contaminants are analysed on a regular basis. Water: Certificates of analysis is performed quarterly

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 50%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
Formulations (w/w) were prepared daily immediately prior to dosing. Adjustment was made for specific gravity of vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples of week 1 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations). Samples of the lowest concentration were checked for homogeneity and accuracy of preparations in week 3.
- Sample procedure: The formulations were stirred during the sampling. Duplicate samples for the determination of the concentrations and stability were taken at 50% height of the formulation. For the determination of the homogeneity, duplicate samples were taken at 90, 50, and 10% height of the formulation.
- Sample pre-treatment: The samples (386-595 mg) were weighed accurately into volumetric flasks (50-100 mL). The flasks were filled up to the mark with mobile phase. If necessary, the solutions were further diluted with mobile phase to obtain concentrations within the calibration range. All sample dilutions were measured in duplicate.
- HPLC conditions: Column: LiChrosper 100 RP-18, 125 x 4 (I.D.) mm; dp = 5µm (Merck Germany); Mobile phase: 80/20 (v/v) acetonitrile/Milli-Q water; Flow: 1mL/min; Detection: UV at 214 nm; Injection volume: 10 µL.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study.

Examinations

Observations and examinations performed and frequency:
- Mortality/Viability: Twice daily
- Clinical signs: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on days 8, 15, 22 and 28, this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales: Maximum grade 4: grading slight (1) to very severe (4); Maximum grade 3: grading slight (1) to severe (3); Maximum grade 1: presence is scored (1).
- Functional Observations: During week 4 of treatment, the following tests were performed on all animals: hearing ability, pupillary reflex, static righting reflex, grip strength (Score 0 = normal/present; Score 1 - abnormal/absent); activity test (based on hourly data per animal, using a computerised motor activity monitoring system, Pearson Technical Services , Debenham, Stowmarket, England)
- Body weight: On days 1, 8, 15, 22 and 28
- Food consumption: Weekly
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
- Clinical laboratory investigations: Blood samples were collected under light ether anesthesia immediately prior to scheduled post mortem examination, between 7.30 and 9 .30 a.m. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for hematological parameters (0.6 mL), with citrate for clotting tests (1.0 mL) and untreated tubes for clinical biochemistry parameters (>2.0 mL).
-- Haematology: The following hematology parameters were determined from blood prepared with EDTA as an anti-coagulant: Erythrocyte count/RBC, Haemoglobin/HB, Haematocrit/HCT, Mean corpuscular volume/MCV, Mean corpuscular haemoglobin/MCH,
Mean corpuscular haemoglobin concentration/MCHC, Platelet count, Red cell distribution width/RDW, Total leucocyte count/WBC, Differential leucocyte count/SEG (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes. The following hematology parameters were determined from blood prepared with citrate as an anti-coagulant: Prothrombin time/PT, Partial thromboplastin time/PTT.
-- Clinical biochemistry: Alanine aminotransferase/(ALAT/GPT), Aspartate aminotransferase/(ASAT/GOT), Bilirubin (total), Cholesterol (total), Creatinine, Glucose, Protein (total), Protein (albumin), Alkaline phosphatase (ALP), Sodium, Potassium, Chloride, Calcium, Phosphorus

Sacrifice and pathology:
- Necropsy: All animals surviving to the end of the observation period were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Photographs were taken from unusual findings noted during macroscopic examination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, Aorta, Brains, Caecum, Cervix*, Clitoral gland*, Colon, Duodenum, Epididymides, Eyes with optic nerve and Harderian gland*, Female mammary gland area*, Femur including joint*, heart, Ileum, Jejunum, Kidneys, Larynx*, Exorbital Lacrimal gland*,Liver, Lung , infused with formalin, Lymph nodes - mandibular , mesenteric, Nasopharynx*, Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland*, Prostate gland, Rectum, Salivary glands – mandibular – sublingual*, Sciatic nerve, Seminal vesicles*, Skeletal muscle*, Skin*, Spinal cord - cervical , midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, Tongue*, Trachea, Urinary bladder, Uterus, Vagina*, All gross lesions.
- Organ weights: The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus
- Histotechnology: All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxyl in and eosin. Additional liver sections were stained by PerIs' method.
- Histopathology: Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, as well as all gross lesions of all animals were examined by a pathologist. Based on the treatment related morphologic changes, liver, spleen, uterus, ovaries, adrenal glands and thymus were also examined from all rats of the intermediate dose groups. All abnormalities were described and included in the report. Tissues with an asterix were not examined as there were no signs of toxicity or target organ involvement.

Statistics:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Alopecia was noted in the majority of animals receiving 50 mg/kg bw/day. Hunched posture, piloerection and emaciation were noted in addition in the majority of females of this dose group. One female showed a wound, scabs and swelling at the tail, most likely caused by trauma. A hunched posture was noted on a few days only in one female receiving 15 mg/kg bw/day. Excessive salivation was mainly observed in treated animals in a dose-related manner. Rales were also observed in some treated animals. These signs are often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or the taste of the test substance. Therefore, these findings were considered not to be a sign of systemic toxicity. Incidental findings that were noted included scabs and alopecia noted among control rats. These findings were considered to be within the normal range of biological variation for rats of this age and strain.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At a dose level of 50 mg/kg bw/day, body weight loss and/or reduced body weight and body weight gain were noted in females from day 8 onwards and in males from day 15 onwards. Body weight gain was (slightly) reduced in females treated at 5 or 15 mg/kg bw/day on day 22 and/or day 28. Body weights and body weight gain of other treated animals remained in the same range as controls over the 4-week study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A reduced absolute food consumption was noted in females treated at 50 mg/kg bw/day, throughout the treatment period. After allowance for body weight, food consumption among these females was reduced mainly during the first week only. There were no differences in food consumption before or after allowance for body weight between other treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At a dose level of 50 mg/kg bw/day, haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin were decreased in males and females and mean corpuscular haemoglobin concentration and total leucocyte count were decreased in females only. Red cell distribution width was increased in males and females of this dose. Haemoglobin, haematocrit, and mean corpuscular volume were decreased in females treated at 15 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alanine and aspartate aminotransferase activity and total bilirubin levels were increased in females treated at 50 mg/kg bw/day. Protein, sodium and calcium levels were decreased and potassium levels were increased in males of this dose group. The decreased creatinine levels in females treated at 50 mg/kg bw/day were considered to be related to the reduced body weights. At a dose level of 15 mg/kg bw/day, protein and calcium levels were decreased in males. Other values in treated animals achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly low control values (potassium in females) or by chance as no treatment related distribution or corroborative findings in the opposite sex were detected (cholesterol, glucose). Moreover, all mean values remained within the normal range of background data for rats of this age and strain.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The variation in motor activity did not indicate a relation with treatment and no abnormalities were noted in the other functional observation tests.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Autopsy body weights were decreased in males and females receiving 50 mg/kg bw/day. Additional changes in this dose group included an increase in liver: body weight ratios and kidney: body weight ratios (males and females) and decreased thymus and adrenal glands weight (females). All other decreases in organ weights of this dose group were considered to reflect a lower body weight. In the 15 mg/kg bw/day dose group, autopsy body weights of females were reduced. Other statistical significant changes in organ weights in the 5 and 15 mg/kg bw/day dose groups were considered to be of no toxicological relevance as no dose response relationship was detected (adrenals) or as the organs are dependent on body weight and no effects were observed on relative values (liver, kidney).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The livers of all animals treated at 50 mg/kg bw/day showed a discolouration, which was generally described as black-brown, but varied between chocolate-coloured , greenish to black. Other findings in females of this dose group were: red-brown discolouration of the kidneys, greenish discoloured lacrimal glands, pale discolouration and/or reduction in size of the adrenal glands, and a reduction in size of the thymus. Alopecia was observed in both males and females of this dose group. A less intensive, light black-brown discolouration of the liver was observed in all females and three males treated at 15 mg/kg bw/day. In addition, light greenish discoloured lacrimal glands and red-brown discoloured kidneys were also observed among the females. A soft fore brain, and a watery cyst in the ovaries, an enlarged spleen and a pale discoloured pancreas, noted among females, were considered to be within the range of biological variation for rats of this age and strain and not to represent a change of toxicological significance based on the absence of a treatment-related distribution and corroborative findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Primary treatment related microscopic findings were noted in the liver and spleen. Midzonal /centrilobular hypertrophy was seen at slight or moderate degrees in all 50 mg/kg bw/day treated males and females, and at minimal or slight degrees in all female rats treated at 15 mg/kg bw/day. This was accompanied in the 50 mg/kg bw/day females by an increased deposition of iron positive pigment in Kupffer's cells and an increase in hepatocyte mitoses. These findings were considered to have contributed to the discolouration noted in the liver at necropsy. In the spleen there was an increase in the severity of hemosiderin pigment deposition in 50 mg/kg bw/day treated males and in 15 or 50 mg/kg bw/day treated females. In the 50 mg/kg bw/day treated males this alteration was accompanied by a slight increase in hemopoiesis. A range of treatment related findings were seen only in the 50 mg/kg bw/day treated animals and were considered to be secondary in nature, related to retarded growth rate. Included amongst these were atrophy of the adrenal glands seen in nine animals, thymus atrophy in eight, reduced numbers of corpora lutea in the ovaries and diestrus epithelium in the uterus of three. Atrophy of the lacrimal glands was seen in all females treated at 50 mg/kg bw/day and in three females treated at 15 mg/kg bw/day. These findings were the microscopic correlates to the gross observations on these organs. There was no microscopic correlate to the discolouration noted in the kidneys of 15 or 50 mg/kg bw/day treated females at necropsy. This may have been due to passive colouration by the compound or metabolites thereof. The remainder of the microscopic findings recorded were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Test substance formulations in propylene glycol formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed mean values of 92%, 94% and 98% of nominal for groups 2, 3 and 4 (5, 15 and 50 mg/kg bw), respectively, which was considered to represent an acceptable level of accuracy for formulations of this type.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion