Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 14, 1994 - July 19, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
1981
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1992
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Test system: Rat, HanIbm (WIST SPF).
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, 4414 Fuellinsdorf/Switzerland.
- Age at delivery: 6 weeks.
- Weight at study initiation: males at 134.54 - 178.89 g; females at 113.22 - 148.01 g.
- Housing: individually in Makrolon type-3 cages (22x37x15 cm) with standard softwood bedding ("lignocel", Schill AG, CH-4132 Muttenz)
- Diet: pelleted standard Kliba 343, Batch 76/94 rabbit maintenance diet ("Kllba", Klingentalmueble AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap water from Itingen, ad libitum. A bacteriological, chimical and contaminant analyses were performed.
- Acclimation period: from January 14, 1994 to January 20, 1994 under laboratory conditions. Only animals without any visual signs of illness were used for the study.
- Identification: by unique cage number and corresponding ear tatoo.
- Randomization: computer-generated random algorithm.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10-15 ACH
- Photoperiod: 12 hours fluorescent light / 12 hours dark; music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
Test article was weighed into a glass beaker on a tared Mettler PM 480 balance and the vehicle was added. The mixture W/v was prepared using ahomogenizer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
Dose volume: 10 ml/kg bw per treatment day.

Concentration, homogeneity and stability of the test article/vehicle mixtures were determined during acclimatization. Further samples for analysis were taken during week 3 of the test and subsequently analysed.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females x dose x group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on data from acute oral toxicity study performed by the sponsor and a 5-day dose-range finding study (project no. 372420) in which the test item was administered orally by gavage to rats.

Examinations

Observations and examinations performed and frequency:
BODY WEIGHT
- Time schedule for examinations: of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION
The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PM 4800 balance connected to the computer.

OPHTHALMOSCOPIC EXAMINATION
At 4 weeks; a description of any abnormally was recorded. 8 - 50 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine BETA 200 Ophthalmoscope.

HAEMATOLOGY and CLINICAL BIOCHEMISTRY
Blood samples for haematology and clinical biochemistry were collected from all animals under light ether anaesthesia. The animals were fastedin metabolic cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected between the hours of 07.00 and 08.30 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-haematocrit glass capillary tube.
- Parameters haematology: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobinconcentration, platelet count, reticulocyte count, reticulocyte fluorescence ratios, nucleated erythrocytes, heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, activated partial thromboplastin time time.
- Parameters clinical biochemistry: glucose, urea, creatinine, bric acid, bilirubin, total, cholesterol, total triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyltransferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein, total, globulin, albumin/globulin ratio.

URINALYSIS
Urine was collected during the 18-hours fasting period into a specimen vial.
- Parameters: volume (18-hour), specific gravity, osmolality, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobllinogen, urine sediment.
Sacrifice and pathology:
NECROPSY
All animals were weighed and necropsied and descriptlons of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for approximately 18 hours, but free access to water was provided.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution: adrenals; aorta; bone (sternum, femur); bone marrow (sternum, femur); brain; cecum; colon; duodenum; epididymides; esophagus; eyes w1th optic nerve and Harderian gland; fémur including joint; heart; ileum; jejunum; kidneys; larynx; lacrimal gland, exorbital; liver; lung infused with formalin; lymph nodes, mandibular, mesenteric; mammary gland area; nasal cavity; ovaries; pancreas; pituitary gland; prostate gland; rectum; salivary gland, (mandibular, sublingual); seminal vesicles; sciatic nerve; skeletal muscle; skin; spinal cord, (cervical, midthoracic, lumbar); spleen; stomach; testes; thymus; thyroid incl. parathyroid gland; tongue; trachea; urinary bladder infused with formalin; uterus; vagina and gross lesions.

HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver, lungs, spleen, stomach and testes collected at terminal sacrifice from the animals, of the control and high-dose groups were examined by a pathologist. The same applied to all gross lesions from all rats. In addition, the spleen was histopathologically examined also in the animals of the intermediate groups. All abnormalities were described and included in the report.
Statistics:
The following statistical methods were used to analyse the body weights, food consumption, organ weights, all ratios and clinical laboratory data.
- When 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 between the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The Fisher's exact test was applied to the ophthalmoscopy data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, one male and one female animal showed ruffled fur on days 2 and 3 or between days 2 and 8 of the treatment period, respectively. Although, this finding was not observed in the other 8 animals of this group, it was considered to be a slight reaction of the animals to treatment with the test article.
At 200 and 1000 mg/kg, all animals had red discoloration of the faeces. This finding was considered to be caused by a passive coloration of the faeces by the red test article but without any toxicological significance.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the course of this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In comparison with the control values, no test article related or statistically significant differences were noted in the body weight development of the animals in all dose groups (50, 200 and 1000 mg/kg).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The differences in food consumption between the control animals and the animals of the dose groups (50, 200 and 1000 mg/kg) were not considered to be correlated to the treatment with the test article. The statistically significant increase in food consumption noted for the group 2 (50 mg/kg) females between days 8 and 22 of treatment period was considered to be incidental because no dose relationship was evident in the other two dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment period, no test article related abnormal findings were noted at ophthalmoscopic examination.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the assessment of haematological data the following effects were recorded in rats of groups 3 (200 mg/kg) and/or 4 (1000 mg/kg) at termination of the treatment:
- Slightly decreased haemoglobin (HB) concentration by 6 % on average in males of group 4.
- Moderately increased reticulocyte count (relative/absolute) by 79 to 74 % on average in males and 127 to 115 % In females of group 4 as well as a slight increase by 29 to 34 % in males and 46 to 47 % in females of group 3. Although the relative increase noted in group 3 did not attain statistical significance, the increase is considered to be treatment-related.
- Slightly increased HFR reticulocyte fluorescence ratio by 113 % and decreased LFR fluorescence ratio by 18 % on average in males of group 4.
- Moderately Increased methemoglobin (MET-HB) concentration by 315 % on average in males and 336 % in females of group 4, and slightly increased by 146% in males and 144% in females of group 3. The increase in the group 3 females did not attain statistical significance, nevertheless, the increase is considered to be treatment-related.
The findings noted in the haematological parameters were considered to be test article related and suggest methemoglobinemia with an increase in hemopoietic activity as indicated by the increase in reticulocytes.
All other differences in the results of the hematology parameters were considered to be incidental and unrelated to the treatment, and of normal biological variation for rats of this strain and age.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the assessment of clinical biochemistry data the only relevant finding to be noted at termination of the treatment was a moderate increase in the total bilirubin concentration by 108 % on average in males and 103 % in females of group 4 (1000 mg/kg) as well as a slight increase by 41 % in males and 62 % in females of group 3 (200 mg/kg). This finding was considered to be test article related and suggests a greater erythrocyte turnover (hemoglobin liberation). This was also supported by the hematological and histopathologlcal findings in these animals.
All other differences in the results of the clinical biochemistry parameters were considered to be incidental and unrelated to the treatment, and of normal biological variation fop rats of this strain and age.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis data indicated no changes of toxicological significance at termination of the treatment, however, the following alterations were noted when compared to the controls:
- Deep yellow urine pigmentation in two females of group 2 (50 mg/kg), in two males of group 3 (200 mg/kg) and in one male and one female of group 4 (1000 mg/kg). Furthermore, a light brown urine discoloration was noted in one female of group 3 and three females of group 4, and a brown discoloration in one female of group 3 and in one male and one female of group 4.
- Marked increase in urine bilirubin (scores) in both sexes of all treated groups.
The abnormally deep yellow and light brown to brown urine pigmentation was considered to be test article related, whereas the higher bilirubin scores suggest false positive reactions due to interference of the test article or metabolites thereof with the reagent test-strip reaction. This was also supported by the f act that "Ictotest" used to confirm positive bilirubin reactions was found to be negative in all cases.
In addition, the observation was not supported by any underlying morphological findings which would suggest disturbed bilirubin metabolism, liver dysfunction or biliary obstruction. It may be noted that normally, urine does not contain any bilirubin. Free or unconjugated bilirubin which results from the breakdown of hemoglobin is not water soluble, is bound to plasma proteins and is not excreted in the urine, whereby conjugated bilirubin (bilirubin glucuronide) which is water soluble, is excreted in the urine.
All other differences in the results of the urinalysis parameters were considered to be incidental and unrelated to the treatment, and of normal biological variation for rats of this strain and age.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The spleen weight was increased in the animals of the highest dose group. This finding was more obvious in the female animals. In the male animals only the spleen/body weight ratio attained statistical significance, whereas in the females also the absolute value and the spleen/brain weight ratio were statistically significantly increased from the respective control values.
This finding was considered to be test article related and correlated with the clinical biochemistry investigations and histopathological data and indicate the spleen as a target organ.
In addition, at 1000 mg/kg, statistically significantly increased relative liver weight was noted for the female animals. This finding was only of marginal nature and was not confirmed neither in the respective male animals nor any histopathological correlate was seen. However, In consideration that also the test article related findings In the spleen were more pronounced in the females than in the males, this finding could be treatment related and could respect a reaction due to the Increased metabolic charge of the liver.
The statistically significantly reduced absolute spleen weight in the group 2 (50 mg/kg) males was considered to be correlated with the reduced terminal mean body weight of these animals and had no correlation with the findings noted in the spleen of the highest dose group animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At terminal necropsy no test article related abnormal findings were noted. The type, incidence, severity and organ distrlbution of the findings noted, did not distinguish the control from the treated animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At 1000 mg/kg, slightly increased splenic extramedullary haematopoiesis was observed In 3 males and 1 female. This finding was treatment related and was in
accordance with the data of the clinical laboratory investigations and with the organ weight analysis.
There were no other treatment related findings. A varlety of other minor degenerative and inflammatory lesions were observed which fell withln the expected range for rats of this age.
Details on results:
At 1000 mg/kg, 2 animals had ruffled fur in some days during the first part of treatment. Although this observation was not recorded in the other animals of this dose level, this finding was considered to be test article related.
At 1000 mg/kg, the assessment of hematological and clinical biochemistry data as well as the organ weight analysis and histopathological examination indicated the spleen as target organ. The hematological data showed methemoglobinemia with an increase in hemopoietic activity as indicated by the increase in reticulocytes. Increase total bilirubin concentration suggests a greater erythrocytes turnover (hemoglobin liberation). These findings were supported by an increase of the spleen weight and by the increased splenic extramedullary haematopoiesis observed at histopathological examination.
In addition, at 1000 mg/kg, increased relative liver weight was noted for the female animals, only. This finding was without any histopathological correlate.
However, in consideration that the test article related findings which indicated the spleen as target organ were generally more pronounced in the females than in the males, a treatment related effect on the liver of the females in form of a reaction due to overload metabolic activity could not be ruled out.
The histopathological examination of the spleen at 50 or 200 mg/kg body weight/day did not reveal any test article related lesions. The hematology and clinical biochemistry data in the 200 mg/kg animals indicated similar findings as in the highest dose group and were therefore considered to be dose related effects.
At 50 mg/kg, no test article related effects were evident.

Effect levels

Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
By the following experiment, the NOEL (28 d) of the test substance was determined to be 50 mg/kg bw for males and females rats, when administrered orally by gavage.
Executive summary:

The test was performed according to OECD 407 (1981).

The test substance was administered daily by gavage to Wistar rats of both sexes at dose level s of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

The following parameters were evaluated: clinical signs, food consumption and body weights, ophthalmoscopy examinations, haematology, clinical biochemistry, urinalysis, organ weights, macroscopic and microscopic findings.

Mortality

No deaths occurred during the course of this study.

Clinical Signs

In 20 % of the animals at 1000 mg/kg, ruffled fur was noted in some days during the first part of the treatment period.

In addition passive coloration of the faeces by the red test article was noted in all animals at 200 and 1000 mg/kg. This finding was considered to be without any toxicological significance.

Food Consumption And Body Weights

Up to and including the highest dose level of 1000 mg/kg, there were no effects on the food consumption or body weight gain of the animals.

Ophthalmoscopic Examinations

No test article related abnormal changes were noted.

Haematology

The assessment of haematological data indicated the following effects in rats of groups 3 (200 mg/kg) and/or 4 (1000 mg/kg) at termination of the treatment:

- Slightly decreased hemoglobin (HB) concentration in males of group 4, slightly to moderately increased reticulocyte count (relative/absolute) in both sexes of groups 3 and 4, slightly increased HFR and slightly decreased LFR reticulocyte fluorescence ratio in males of group 4, and slightly to moderately increased methemoglobin (MET-HB) concentration in both sexes of groups 3 and 4.

The findings were considered treatment-related and suggest methemoglobinemia with an increase in hemopoietic activity as indicated by the increase in reticulocytes.

Clinical Biochemistry

In the assessment of clinical biochemistry data the only change of note was a slightly to moderately increased total bilirubin concentration in both sexes of groups 3 (200 mg/kg) and 4 (1000 mg/kg) at termination of the treatment. This finding was considered to be treatment-related and suggests a greater erythrocyte turnover (hemoglobin liberation).

Urinalysis

Urinalysis data at termination of the treatment indicated no changes of toxicological significance. The only changes noted were a deep yellow urine pigmentation in two females of group 2 (50 mg/kg), in two males of group 3 (200 mg/kg) and in one male and one female of group 4 (1000 mg/kg). In addition, a light brown urine discoloration was noted in one female of group 3 and three females of group 4 as well as a brown discoloration in one female of group 3 and in one male and one female of group 4. Also noted was a marked increase in urine bilirubin (scores) in both sexes of all treated groups, however, this latter finding suggests false positive reactions, the apparent result of interaction of the test article or metabolites thereof with the reagent test-strip reaction. This was also supported by the fact that "Ictotest” used to confirm positive bilirubin reactions was found to be negative in all cases nor was this observation supported by any underlying morphological findings which would suggest disturbed bilirubin metabolism, liver dysfunction or biliary obstruction.

Organ weights, organ to body weight and organ to brain weight ratios

At 1000 mg/kg, increased spleen weight was in particular for the female animals. This finding was considered to be test article related and correlated with the clinical biochemistry investigations and histopathological data and indicate the spleen as a target organ.

In addition, at 1000 mg/kg, increased relative liver weight was noted for the female animals. This finding was without any histopathological correlate.

However, in consideration that also the test article related findings which indicated the spleen as target organ were more pronounced in the females than in the males, this finding could be treatment related and could respect a reaction due to the increased metabolic charge of the liver.

Macroscopic and microscopic findings

At terminal macroscopic examination no test article related abnormal findings were noted. At 1000 mg/kg, increased splenic extrainedullary haematopoiesis was observed. This finding was treatment related and was in according with the data of the clinical laboratory investigations and with the organ weight analysis.

Based upon the results obtained in the study, the NOEL (28 d) of test item was considered to be 50 mg/kg bw for male and females rats when administered orally by gavage.