Pentasodium 2,2'-((2,4-diamino-5-(4-methoxy-2-sulfophenylazo)-1,3-phenylene)bis(azo))bis(5-(2-(sulfonatooxyethyl)sulfonyl)benzenesulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 5, 2006 to Aug. 22, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(weight of heart was not determined)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

(weight of heart was not determined)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: OPPTS 870.3050

Deviations:

yes

Remarks:

(weight of heart was not determined)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

475-300-1

EC Name:

-

Cas Number:

910030-59-0

Molecular formula:

Hill formula: C29H25N8Na5O22S7 CAS formula: C29H30N8O22S7.5Na

IUPAC Name:

Pentasodium 2,2'-((2,4-diamino-5-(4-methoxy-2-sulfophenylazo )-1,3-phenylene)bis(azo))bis(5-(2-(sulfonatooxyethyl)sulfony l)benzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

see below

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI) BR rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi Coop Ltd. 1032 Budapest, Cserkesz u. 91.
- Hygienic level at arrival: SPF
- Animal health: Only healthy animals were used for the study.
- Age at study initiation: Young adult rats, 6-8 weeks old
- Body weight range at treatment: Male: 176 - 199 g; Female: 158 - 182 g
- Housing: Type III polypropylene/polycarbonate, Five animals /cage
- Bedding: Laboratory bedding, changed together with the cages twice a week
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum
- Water: Tap water from 500 mL bottle, ad libitum
- Acclimation period: Female: 6 d
- Animal identification: The individual identification was performed by tattoo numbers on the tail. The numbers were given on the basis of LAB International Research Centre Hungary Ltd.'s master file, for each animal allocated to the study. The boxes were marked by identity cards, with information about study code, sex, dose group, cage number and individual animal numbers.
- Randomisation: Animals were sorted according to the body weight and allocated to the test groups taking animals from each weight range group on the daybefore the start of the treatment period. The results of the randomisation were checked according to the actual body weights assuring an acceptable homogeneity and variability among the groups.

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30 - 70%
- Air changes: 8-12 air exchanges/hour by central air-condition system
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: July 11, 2006 to August 22, 2006

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method of administration: Gavage
PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated in different concentrations prepared with distilled water. Formulations were prepared daily during working days and beforehand for weekends. Test substance was stable at the applied concentrations for 24 h at room temperature and in refrigerator (5±3°C) for 4 d.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 6.25 mg/mL, 25 mg/mL and 100 mg/mL
- Treatment Volume: 10 mL/kg bw
- Lot/batch no. of vehicle: 3490306
- Expiry Date of vehicle: March, 2009
- Storage of vehicle: At room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of concentration of dosing formulations was conducted on Days 3 and 23. All the measured concentrations varied in the range of 91 and 99% of the nominal concentrations.

Duration of treatment / exposure:

28 d

Frequency of treatment:

once daily (i.e., 7 d/week for a period of 28 d)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

based on test material, analytically verified

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

based on test material, analytically verified

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

based on test material, analytically verified

No. of animals per sex per dose:

Five/sex: 62.5 and 250 mg/kg bw/day
Ten/sex: 0 and 1,000 mg/kg bw/day (incl. five/sex in recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Justification of doses: The doses were chosen on the basis of the results of repeated dose toxicity studies of compounds with similar chemical structure.

Experimental design
-The concentration of the test substance formulations and the number of animals were as follows:

GROUP Dose mg/kg bw/day Concentrationmg/mL No. of Animals
male female
Group 1 Control 0 5+5* 5+5*
Group 2 62.5 6.25 5 5
Group 3 250 25 5 5
Group 4 1,000 100 5+5* 5+5*
* Animals for recovery groups.

Randomisation
- Animals were sorted according to the body weight and allocated to the test groups taking animals from each weight range group on the day before the start of the treatment period. The results of the randomisation were checked according to the actual body weights assuring an acceptable homogeneity and variability among the groups.

Treatment Volume
- A constant treatment volume of 10 mL/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent body weight of the animals.

Procedure
- Animals were dosed with the single dose of the test substance by gavage daily 7 d each week for a period of 28 d. Animals in the control group were treated with the vehicle and handled in an identical manner to those in the test groups. Treatment was carried out at similar times (± 2 h) in the morning each day-The first treatment day was considered as day 0. Animals were not treated on the day of necropsy.
- Duration of treatment: 28 d
- Animals of the recovery groups were not treated from 28 d. Daily and detailed weekly clinical observation, weekly body weight and food consumption measurements were conducted for a 14 d period. All recovery animals were processed on the same way as animals at termination of the treatment.

Examinations

Observations and examinations performed and frequency:

1. Clinical Observations
- Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
- General clinical observations were made once a day, after treatment at approximately the same time.
-Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once a week thereafter.
-Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (i.e., lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.
-Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on all animals in the fourth exposure week (i.e., 27 d). General physical condition and behaviour of animals were tested. A modified Irwin test was performed.

2. Body Weight Measurement
-Body weight was measured on Day 0 (beginning of treatment), then weekly with a precision of 1 g.

3. Food Consumption Measurement
-The food consumption was determined weekly by re-weighing the non-consumed diet with a precision of 1 g.

4. LABORATORY EXAMINATIONS
-Laboratory examinations including haematology and clinical chemistry were conducted terminally and at the end of the recovery period. After an overnight food deprivation (i.e., approximately 16 h), animals were anaesthetised with Euthanyl and blood samples were collected by heart puncture. Three samples were taken from each animal: one for haematology (tubes contained K3-EDTA as anticoagulant), one for determination of blood clotting times (APTT and PT; tubes contained sodium citrate as anticoagulant) and the third one to obtain serum samples (tubes did not contain any anticoagulant) for clinical chemistry.

Haematology
-Haematology parameters evaluated included Red Blood Cell (erythrocyte) count (i.e., RBC), White Blood Cell (leukocyte) count (i.e., WBC), Haemoglobin concentration (i.e., Hgb), Haematocrit (i.e., Hct), Mean Corpuscular (erythrocyte) (i.e., MCV), Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration (i.e., MCHC), Platelet (thrombocyte) count (i.e., Plt), Partial Thromboplastin Time (i.e., APTT), Prothrombin Time (i.e., PT), Differential white blood cell count

Clinical Chemistry
-Clinical chemistry parameters evaluated consisted of Blood sugar conc., Total Bilirubin conc, Urea conc, Cholesterol conc, Creatinine conc., Phosphorus conc., Sodium conc., Potassium conc., Calcium conc, Chloride conc., Total Protein conc., Albumin conc., Alb/glob ration, Aspartate Aminotransferase activity, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -Activity.

Sacrifice and pathology:

Necropsy
-A gross necropsy was performed on each animal just after the blood harvesting for clinical pathology examinations. 1 d after the last treatment (i.e., 28 d), animals were sacrificed under pentobarbital (Euthanyl) anaesthesia. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Any abnormality was recorded with details of the location, colour, shape and size.

- The following organs and tissues were preserved in 10 % formaldehyde solution for histological evaluation:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, testes with epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, lachrymal gland with Harderian gland, seminal vesicle, muscle (quadriceps), sciatic nerve and aorta.

Histopathology
-Histological examinations were performed on the preserved organs or tissues of the animals of the control and high dose groups. In groups 2 and 3, liver and kidneys were also processed histologically.
-The listed organs or their specimens were embedded into paraffin after dehydration. Slides were stained with haematoxylin eosin and examined by a light microscope.

Other examinations:

Organ Weight Measurement
- The following organs were weighed and recorded. Paired organs were weighed together.

With precision of 0.01g: Liver, kidneys, testes, epididymides, thymus, spleen, brain.
With precision of 0.001g: Adrenals

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- haematology
- clinical chemistry
- organ weight

The heterogeneity of variance between the groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a oneway analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of the inter-group differences. Where significant heterogeneity was found, the normal distribution of data by Kolmogorov-Smirnov test was used. In case of not-normal distribution, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was applied. If positive results were obtained, the inter-group comparisons were performed using Mann-Whitney U-test.

Frequency of clinical symptoms, necropsy and histopathological findings and daily mean food consumption were calculated.
At the end of recovery period, the homogeneity of variance between groups was determined by F-test. Depending on the result Poled or Separate variance estimate of the Two-Sample t-test was performed. For data, which was not normally distributed, the data were compared between groups according to Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There was no mortality. Also, there were no test substance related clinical symptoms and no functional observation battery effects.

Mortality:

no mortality observed

Description (incidence):

There was no mortality. Also, there were no test substance related clinical symptoms and no functional observation battery effects.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance related differences in the body weight development between any dose groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance related differences in the mean food consumption between any dose groups

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance related differences in the examined haematological parameters

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance related differences in the examined clinical chemistry parameters

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no relevant test substance related organ weight alterations

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no relevant test substance related macroscopic changes

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no histopathological changes related to the test substance effect detected.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

MORTALITY
-There was no mortality during the study

CLINICAL OBSERVATIONS
Daily and Detailed Weekly Clinical Observations

-There were no clinical symptoms related to the treatment in the control, 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1,000 mg/kg bw/day groups in the course of the 28 d observation period and during the recovery phase.
-The physiological state and behaviour of animals were considered to be normal in the control and in all test substance treated groups at the daily and at the detailed weekly observation.

Functional Observation Battery

Group 1 – Control
-In the male animals, an exaggerated startle reaction (1/10), increased (1/10) or reduced (1/10) transfer arousal, increased (1/10) or reduced spatial locomotion (1/10) and increased (1/10) or reduced touch escape response (1/10) and positional struggle to a marked degree (1/10) were observed.
In the female animals, a decreased transfer arousal (3/10), vigorous spatial locomotion (3/10) and positional struggle to a marked degree (4/10) lack of finger approach (2/10) and finger withdrawal (2/10) were noted.

Group 2 – 62.5 mg/kg bw/day
-In the male animals, increased transfer arousal (1/5), elongated finger approach (1/5) and lack of finger withdrawal (1/5) were noted. In the female animals, reduced transfer arousal (1/5), increased spatial locomotion (1/5), lack of finger approach (1/5) and finger withdrawal (1/5), positional struggle to a marked degree (1/5) and vocalisation (1/5) were observed.

Group 3 – 250 mg/kg bw/day
-In the male animals, reduced transfer arousal (1/5) was observed in one rat.
-In the female animals, an increased locomotor activity (1/5), exaggerated startle reaction (1/5) reduced transfer arousal (1/5), increased spatial locomotion (1/5), lack of finger approach (1/5), vigorous finger withdrawal (1/5) and touch escape response (1/5) were noted.

Group 4 – 1,000 mg/kg bw/day
-In the male animals, increased transfer arousal (3/10), lack of finger approach (2/10) and lack of finger withdrawal (2/10) were found. In the female animals, an increased startle reaction (1/10) and transfer arousal (1/10), an elongated finger approach (1/10), lack of finger approach (1/10) and lack of finger withdrawal (1/10), touch escape response (1/10) and positional struggle to a markeddegree (1/10) were observed.

-In summary: no test substance related clinical symptoms were noted after 28 d administration of test substance. There were also no test substance effects at the functional observation. Changes in locomotor activity, startle reaction, transfer arousal, spatial locomotion, positional struggle, vocalisation, finger approach and finger withdrawal, in touch escape reaction were observed with similar incidences in each group and represent inter individual differences in animal’s behaviour, reactions to different type of stimuli or manipulations.

BODY WEIGHT AND BODY WEIGHT GAIN

Group 2 – 62.5 mg/kg bw/day
-There were no significant differences in the mean body weight and mean body weight gain between the control and 62.5 mg/kg bw/day groups (male and female) during the treatment period.

Group 3 – 250 mg/kg bw/day
-The mean body weight and body weigh gain of male and female animals were similar to the control value during the experiment.

Group 4 – 1,000 mg/kg bw/day
-The mean body weight and body weight gain were comparable to the control value both in male and female animals during the treatment and the recovery periods.

-In summary: Test substance did not influence the body weight development at the applied doses during the 28 d observation period. There were no differences between the experimental groups in the body weight and body weight gain consequently there was no difference at termination of the recovery period.

FOOD CONSUMPTION
-No test substance related changes were observed in the daily food consumption in male and female groups at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1,000 mg/kg bw/day doses. The mean daily food intake was also similar in the control and high dose animals during the recovery period.

HAEMATOLOGY

Group 2 – 62.5 mg/kg bw/day
-In the male animals, all examined haematological parameters were similar to the control value.
-In the female animals, the prothrombin time (PT) was slightly less than the control value. The WBC exceeded the control value.

Group 3 - 250 mg/kg bw/day.
-In the male group, the haemoglobin concentration (HGB), haematocrit (HCT) and white blood cell count (WBC) were slightly above the control value.
-In the female animals, the PT was slightly shorter than the control value. The WBC exceeded the control value.

Group 4 – 1,000 mg/kg bw/day
-In the male animals, platelet count (PLT) and lymphocyte cell count (LY) were slightly higher.
-In the female animals, the PT was slightly less the eosinophyl cell count (EO) was higher than the control value. The WBC exceeded the control value without statistical significance.
-At the end of the recovery period, in the male animals the mean corpuscular haemoglobin concentration (MCHC) was slightly less than the control value.
There were no differences in the female animals.
-In summary: No test substance related effect was found in the examined haematological parameters. The slight differences in some parameters of male rats (HGB, HTC, platelet, white blood cell or lymphocyte counts and MCHC) as well in the female rats (blood clotting times and eosinophyl cell count) were within the normal range (historical data) and these were judged to be independent from the treatment, considering the low magnitude and lack of any dose relevance. The white blood cell differences in female treated groups were relative small were due to the unusually low value of control group.

CLINICAL CHEMISTRY

Group 2 – 62.5 mg/kg bw/day
-The calcium (Ca2+) concentration was higher than the control value in male and in female groups.

Group 3 - 250 mg/kg bw/day.
-In the male animals, slightly higher calcium (Ca2+) level and an elevated total bilirubin (T-BIL) concentration were found.
-In the female animals, calcium (Ca2+) concentration slightly exceeded the control value.

Group 4 – 1,000 mg/kg bw/day
-In the male animals, higher concentrations of calcium and total bilirubin and less enzyme activity of aspartate aminotranspherase (AST) were recorded.
-In the female animals, the calcium and T-BIL concentrations were higher.
-In the recovery group, there were no differences in male groups, the concentration of urea, calcium and phosphorous (Phos) were slightly above the control value in female group.

-In summary: Test substance related differences were observed in measurements of total bilirubin concentrations, which were dose dependent in male groups from 250 mg/kg bw/day. In female animals it was significant only at 1,000 mg/kg bw/day. Clinical pathology and histological examinations did not reveal any related pathological changes, so this single alteration was interpreted as a photometric interference of test substance in the serum with the bilirubin. The statistical differences in calcium concentrations in all groups remained in the normal range and were not considered to be biologically relevant.
The statistically significant differences in some parameters (activity of AST in high dose male animals, concentration of urea and phosphorous in recovery female animals) were considered not related to the treatment because of the singular occurrence or low magnitude of the alteration or lack of any dose relevance or any related pathology changes.

NECROPSY
Group 1 – Control
-In the male animals, pinprick-sized haemorrhages in the lungs (3/5) were noted.
-In the female animals, pinprick-sized haemorrhages in the lungs (3/5) and hydrometra (2/5) were observed.
-In the recovery control group, pinprick-sized haemorrhages in the lungs (1/5 male; 2/5 female) and hydrometra (1/5) were seen.

Group 2 – 62.5 mg/kg bw/day
-In the male animals, in the lungs pinprick-sized haemorrhages (3/5) and one side pyelectasis (1/5) were noted.
-In the female animals, pinprick-sized haemorrhages were present in the lungs (3/5).

Group 3 - 250 mg/kg bw/day
-In the male animals, in the lungs pinprick-sized haemorrhages (3/5) and one side pyelectasis (1/5) were observed.
-In the female animals, pinprick-sized haemorrhages in the lungs (4/5) and hydrometra (2/5) were observed.

Group 4 – 1,000 mg/kg bw/day.
-In the male animals, pinprick-sized haemorrhages were noted (3/5) in the lungs. In the female animals, in the lungs pinprick-sized haemorrhages (2/5) were found.
-In the recovery 1,000 mg/kg bw/day group, pinprick-sized haemorrhages in the lungs (3/5 male), one side pyelectasis (1/5 male) and hydrometra (1/5 female) occurred. The left side lobe of lungs was adhered to the thorax in one (1/5) male animal.

-In summary: No test substance related macroscopic findings were found at the necropsy. The haemorrhages in the lungs of animals were caused by the euthanasia procedures and were present with similar incidence in the control and in the test substance treated groups. Pyelectasis is a common disorder in experimental rats. Hydrometra due to the sexual cycle of animals occurred in some female animals of the control and dose groups. The adhesion of lung occurred only in one recovery animal and was considered as individual lesion, which will be cleared up by histological examination.

ORGAN WEIGHT

Group 2 – 62.5 mg/kg bw/day
-There were no significant differences in the examined organ weights between the control and 62.5 mg/kg bw/day groups (male and female).

Group 3 - 250 mg/kg bw/day
-In the male animals, the mean kidney weight relative to the body weight was less than the control value.
-In the female animals, the brain weights relative to the body weight was less and body weight relative to the brain weight was higher than the control value.

Group 4 – 1,000 mg/kg bw/day.
-In the male animals, there were no differences from the control in the examined organ weights at termination of the treatment or the recovery period.
-In the female animals, the brain weights relative to the body weight was less and body weight relative to the brain weight was higher. In the recovery period the thymus weight was slightly less than the control value.

-In summary: No test substance related differences were noted in the weighed organs of test groups. The small differences in weights of kidneys, brain and thymus were considered independent from the test substance because the magnitude of all these differences was small and the absence of any related clinical pathology or histopathology changes indicated that these statistical significances are not representative of adverse effects.


HISTOPATHOLOGY

Control
-In the male animals, in the lungs focal haemorrhages (2/5) and alveolar emphysema (2/5), in the liver focal proliferation of cells belonging uterus dilatation (2/5) were noted.

-In the control recovery group, alveolar emphysema (1/5 male and 1/5 female) and focal haemorrhages (2/5 male, 1/5 female) and foamy cells (1/5 female) were found in the lungs. Uterus dilatation (1/5) were also observed.

Group 2- 62.5 mg/kg bw/day
-One side pyelectasia was noted in one male animals (1/5). There were no histopathological findings in the liver.
-There were no histopathological findings in the liver and kidney of female animals.

Group 3- 250 mg/kg bw/day
-One side pyelectasis was observed in one of male animals (1/5). In the female animals, focal proliferation of MPS-cells was detected in the liver (1/5).

Group 4-1,000 mg/kg bw/day
-In the male animals, in the lungs focal catarrhal pneumonia (2/5) focal haemorrhages (1/5) and focal proliferation of cells belonging to the mononuclear phagocyte system in the liver (1/5) were observed.
-In the female animals, alveolar emphysema and focal haemorrhages were observed in the lungs in one rat (1/5).
In the recovery 1,000 mg/kg bw/day group, in the lungs focal catarrhal pneumonia with focal pleuritis (1/5 male), alveolar emphysema (2/5 male), focal haemorrhages (2/5 male) and foamy cells (1/5) were found. One side pyelectasia was present in one male animal (1/5). In the liver, focal proliferation of cells belonging to the mononuclear phagocyte system (1/5 male and 1/5 female) were observed.

-In summary: In the organs of animals subjected to histopathological examination no test substance related lesions were detected.

-The alveolar emphysema and focal haemorrhage in the lungs occurred in control and treated animals with the some incidence and were considered as consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguination.

-Focal catarrhal pneumonia occurred in three male animals at 1,000 mg/kg bw/day, accompanied with focal pleuritis in one case (No.: 882). The focal catarrhal pneumonia probably was an individual disorder, originated from infection. However, the high dose of test substance may have an influence on the decrease of general resistance of lungs in some animal to the local infections.

-The focal alveolar histiocytosis occurred in 2 control animals and one animals at 1,000 mg/kg bw/day and was considered as incidental finding. The foamy cells (alveolar histiocytosis) is a common incidental finding in older rats and consists of small collections of alveolar macrophages with abundant foamy (lipid-containing) cytoplasm. These are often sub-pleural or located in the more peripheral regions of the lung.

-The focal proliferation of cells belonging to the mononuclear phagocyte system (MPS) in the liver occurred in control and treated animals, may be considered to be of nutrition-physiologic-metabolic origin.

-The pyelectasia (one side) in the kidney occurred sporadically and could be considered as individual disorder.

-The dilatation of the uterine horns in some female animals - in connection with the hydrometra - is a slight neuro-hormonal phenomenon in connection with the sexual function of the inner genital organs.

-There was no morphological evidence of acute or subacute injury of alimentary tract, the cardiovascular system, the immune system, the haematopoietic system, the skeleton, the muscular system, or the central or peripheral nervous system. The structure and the cell morphology of the endocrine glands were similar at the control and treated animals.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

2

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Deviation from the Guidelines:

-Concerning: Organ weight measurement

-Deviation: Weight of heart was not determined

-Reason for deviation: Technical mistake

-Presumed effect on the study: This deviation was not considered to have affected the integrity of the study.

Applicant's summary and conclusion

Conclusions:

Based on absence of treatment related adverse effects, the NOEL were established at the highest tested dose of 1,000 mg/kg bw/day.

Executive summary:

28 d oral (gavage) repeated dose toxicity study was conducted in CRL:(WI) BR rat according to OECD Guideline 407, EPA OPPTS 870.3050 and EU Method B.7 in compliance with GLP with deviation which did not impact the study results..

 

Groups of male and female rats received the test substance at dose levels of 0, 62.5, 250 or 1,000 mg/kg bw/day by oral gavage for a period of 28 d. 14 d recovery groups (controls and high dose animals) were also included in the study.

 

There were no unscheduled deaths throughout the study. No test substance related clinical symptoms and functional observation battery effects were observed in any of the treatment group Also, no effect was observed on body weight and food consumption. Likewise, no test substance related differences were observed on organ weight and haematological and clinical chemistry parameters. Furthermore, no test substance-related gross pathology and histopathological findings were detected.

 

Based on absence of treatment related adverse effects, the NOEL was established at the highest tested dose of 1,000 mg/kg bw/day.