Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)

Repeated dose toxicity: oral

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:

1995

Report date:

1995

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)

Version / remarks:

May 12, 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 31, 1992.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)

EC Number:

419-210-2

EC Name:

Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)

Cas Number:

178452-71-6

Molecular formula:

C66H48CrN16Na5O28S6

IUPAC Name:

chromium(3+) pentasodium bis(7-{4-[2-(1-butyl-5-cyano-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridin-3-yl)diazen-1-yl]benzenesulfonamido}-2-[2-(5-nitro-2-oxido-3-sulfonatophenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hanlbm

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: BRL, Blological Research Laboratories Ltd. Wolferstrasse 4, 4414 Fuelllnsdorf/Switzerland
-Age at study initiation: 6 weeks
-Weight at study initiation:
Males: 138.9 - 152.0 g
Females: 114.0 - 126.7 g
-Housing: Individually in Makrolon type-3 cages with autoclaved standard softwood bedding (Lignocer. Schill AG, CH-4132 Muttenz).
-Diet: Pelleted standard Kliba no. 343, Batch no. 84/94, rat maintenance diet ('Kliba', Klingentalmueble AG, CH-4303 Kaiseraugst) ad libitum.
-Water: Community tap-water from Füllinsdorf was available ad libitum.
-Acclimation period: From 01-FEB-1995 to 07-FEB-1995 under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
-Temperature: 20-21.5 °C
-Humidity: 46 - 68 %
-Air changes: 10-15 air changes per hour
-Photoperiod: 12 hours cycle dark/light
-Other: music during the light period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item 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 a homogenizer. Homogeneity of the test item in the
vehicle was maintained during treatment using a magnetic stirrer. Concentratlon, homogeneity and stability of the test item were determined in samples taken during acclimatization and during week 3 of the treatment.

VEHICLE
-Amount of vehicle: 10 ml/kg body weight per treatment day.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC method

Details on analytical verification of doses or concentrations:

SAMPLE PREPARATION AND STORAGE
Test item were prepared and about 2 g (weighed to the third decimal place) was weighed into a 100 ml volumetric flask. These samples for analysis of concentration and homogeneity were collected and immediately deepfrozen delivered to the analytical laboratory, where they were worked-up and analyzed by HPLC. Samples for analysis of stability were collected after storage of the test item at room temperature for two hours. Then. they were delivered to the analytical laboratory followed by sample work-up and analysis by HPLC. If necessary, worked-up sample solutions were stored in the refrigerator prior to analysis by HPLC.

ANALYTICAL PROCEDURE
-Standard Solutions
A stock solution of the test item in solvent A* with a concentration of 0.2 mg/ml was prepared as follows: 20 mg of the test item was weighed into a 100 ml volumetric flask and dissolved in solvent A* by treatment in an ultrasonic bath. Next, the volumetric flask was filled to volume with solvent A*. Finally, various standard solutions were prepared by respective dilution of this stock solution with solvent A* to yield concentrations in the range from 20 µg/ml to 200 µg/ml. These standard solutions were used to calibrate the HPLC.
-Analysis of Samples
About 70 ml of solvent A* was added to the delivered samples and dissolution was achieved by treatment in an ultrasonic bath. Then the volumetric flask was filled to volume with solvent A*. Depending on the dose group, the tatter sample solution was further diluted with solvent A* to yield a concentration within the calibration range. Finally, a 10 µl aliquot was quantified by HPLC.

Solvent A*: 1000 ml of bi-distilled water with 10 ml of 1 M sodium hydroxide solution

HPLC-DETERMINATION
-Apparatus:
Merck L6200 pump
Merck L-4000 photometer
Shimadzu C-R4A integrator
Merck 655A 40 sampling unit
-Column: Lichrospher RP 18; 5 µm; 125 x 4.6 mm
-Temperature: Room temperature
-Eluent A 40%(v/v):
Acetonitrile 15 % (v/v)
Bi-distilled water 85 % (v/v)
Tetrabuthylammoniumbromide 3.2 g/l
-Eluent B 60 % (v/v):
Acetonitrile 100 % (v/v)
Tetrabuthylammoniumbromide 3.2 g/l
-Flow: 1.0 ml/min
-Detection: UV, 254 nm
-Injection volume: 10 µl

CALIBRATION CURVE
Standard concentration (Y)
(mg/ml) Peak area (X) (counts)
before samples after samples
20 115989 120151
50 292316 269353
100 604250 562776
200 1262192 1170714

Y = 3.35 + 1.62 E-4 • X (R^2 - 0.996)

where
Y = µg/ml of test item injected sample
X = peak area of injected sample in counts

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week for a total of 28 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- Dose groups 1 and 4 (0 and 1000 mg/kg bw): 10 males; 10 females per dose
- Dose groups 2 and 3 (50 and 200 mg/kg bw): 5 males; 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: the choice of dose levels was based upon data from acute oral toxicity study and a 5-day dose-range finding study.

Examinations

Observations and examinations performed and frequency:

MORTALITY / VIABILITY AND CLINICAL SIGNS
-Time schedule: once daily.

FOOD CONSUMPTION
-Time schedule: 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 RCC computer.

BODY WEIGHT
-Time schedule: weekly and twice during week 4 of treatment and week 2 of the recovery period.


OPHTHALMOSCOPIC EXAMINATION:
-Time schedule: twice (at 4 weeks on all survlvlng animals and at 6 weeks on all survlvlng recovery animals)
A description of any abnormality was recorded. 10 - 90 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 (Eisenhut Vet. AG. CH-4123 Allschwil).

HAEMATOLOGY
-Time schedule for collection of blood: twice, after 4 weeks and after 6 weeks
-Anaesthetic used for blood collection: Yes
-Parameters:
Erythrocyte count (RBC): Hydrodynamic focusing electric resistance detection and Sysmex (TOA) E-4000 Multi-Parameter Automated Haematology Analyser
Hemoglobin (HB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Calculated value: HB/HCT - Sysmex (TOA) E-4000 Multi-Parameter Automated Haematology Analyser
Platelet count (PLATELETS)
Reticulocyte count (RETIC.)
Reticulocyte fluorescence ratios (HFR = high MFR = middle LFR = low)
Nucleated erythrocytes (normoblasts)(NEN)
Heinz bodies (HEINZ-B0D.)
Methaemoglobin (MET-HB)
Total leukocyte count (WBC)
Differential leukocyte count (Diff. WBC Count)
Red cell morphology
Coagulation:
Thromboplastin time (=Prothrombin time) (PT)
Activated partial thromboplastin time (APTT)

CLINICAL BIOCHEMISTRY: Yes
-Parameters checked:
Glucose
Urea
Creatinine
Uric acid
Bilirubin total (BILI. T.)
Cholesterol, total (CHOLEST. T.)
Triglycerides (TRIGL.)
Phospholipids (PHOS.LIPID)
Aspartate aminotransferase (ASAT/GOT)
Alanine aminotransferase (ALAT/GPT)
Lactate dehydrogenase (LDH)
Creatine kinase (CK)
Alkaline phosphatase (ALP)
Gamma-glutamyltransferase (G-GT)
Calcium
Phosphorus
Sodium
Potassium
Chloride
Albumin
Protein, total
Globulin
Albumin/Globulin ratio (A/G RATIO)

URINALYSIS:
Urine was collected during the 18-hours fasting period into a specimen vial.
-Time schedule for collection of urine: twice, after 4 weeks and after 6 weeks
-Parameters:
Volume (18-hour):
Specific gravity (SPEC. GRAV.)
Osmolality
Color
Appearance
pH
Protein
Glucose
Bilirubin
Blood
Urobilinogen (UROBILI.)
Urine Sediment (SED. MICRO.)

Sacrifice and pathology:

GROSS PATHOLOGY
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for approximately 18 hours, but free access to water was provided. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. At the end of the treatment or recovery period the animals
designated according to the necropsy schedule as well as all moribund animals were anesthetized by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of 2.0 ml/kg body weight (equivalent to 320 mg sodium pentobarbitone/kg body weight), weighed and sacrificed by exsanguination.
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 with optic nerve and Harderian gland; femur 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.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland.
The organ to terminal body weight ratios as well as the organ to brain weight ratios were determined.
The determination of the terminal body weight was performed immediately prior to necropsy using an on-line electronic recording system consisting of a Mettler PM 4000 balance connected to a computer system.

HISTOTECNIQUE
All organ and tissue samples, as defined under Histopathology were processed, embedded, cut at an approximate thickness of 4 micrometres, and stained with haematoxylin and eosin.

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 and to all animals which died spontaneously or have to terminated in extremis. 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 and 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.
Statistical significances are denoted in tabulated data by the use of superscripts:
Significantly different from the control group (p < 0.05).
Significantly different from the control group (p < 0.01).
The Fisher's exact test was applied to the ophthalmoscopy data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical signs were noted in any group, except in one male (no.15) of group 2 (50 mg/kg) which developed on test day 5 sedation (grade 3), dyspnea (grade 1) and ruffled fur (grade 2).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male (no.15) of group 2 (50 mg/kg) was killed in extremis. This animal developed on test day 5 sedation, dyspnea and ruffied fur. From the section examined, the cause of moribundity could not be established. All other animals survived the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect on body weight in any group of treated males or females. The body weight development was similar in all groups and no statistically significant differences occurred during the study. The slight step-like decrease in body weight observed in the animals assigned to the recovery groups was attributed to the fasting period which preceded measurement.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In all animals the absolute food consumption was slightly but consistently increased over the treatment period. In group 4 (1000 mg/kg) absolute and relative food consumption was higher compared to the other treatment groups and controls. In female animals of group 4 (1000 mg/kg) the difference in absolute food consumption attained statistically significance (p < 0.01) between days 15-22 and 22-28.
The relative food consumption of all animals decreased during the study period. In group 4 (1000 mg/kg) the difference in relative food consumption attained statistically significance (p < 0.01) in males between days 8-15 and in animals of both sex (p < 0.01) between days 15-22 and 22-28. The overall mean food consumption (absolute and relative) was in the same range for treated animals and controls.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related abnormal findings were noted. The few findings Included persistent pupillary membrane, persistent hyaloid vessels in vitreous body and corneal opacity. As these findings occurred with similar incidences in all treated groups, they are considered to be unrelated to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The assessment of haematological data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. However, a few minor statistically significant inter-group differences were recorded in rats of group 4 (1000 mg/kg) after 4 weeks of treatment. These were as follows:
-Marginally lower haemoglobin concentration and haematocrit in females (p < 0.05).
-Slightly higher total leukocyte count in males (p < 0.01).
-Slight proportional changes in the differential leukocyte count in males, i.e. a decrease in segmented neutrophils and increase in lymphocytes (p < 0.05).
Following a two week recovery period these findings were no longer statistically significant and comparable to those of the controls.
All other statistical differences in the results of the haematology and clinical biochemistry parameters were considered to be incidental or unrelated to the treatment and of normal biological variation fop rats of this strain and age.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For clinical biochemistry data there were no changes of toxicological significance at termination of the treatment. The only change of note was a slight but statistically significant increase in the creatinine concentration (p < 0.01) and slight but statistically significant decrease in the total cholesterol and phospholipid concentration (p < 0.05 - p < 0.01) in female rats of group 4 (1000 mg/kg) after 4 weeks of treatment. These findings were found to be reversed and comparable to those of the controls following a two week recovery period.
All other statistical differences in the results of the clinical biochemistry parameters were considered to be incidental or unrelated to the treatment and of normal biological variation for rats of this strain and age.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in the physical properties, i.e. colour and appearance, and in the chemical test-strip-reaction for occult blood were recorded in rats of the treated groups at termination of the treatment when compared to the controls.
These findings were characterized as follows:

URINE PIGMENTATION
Group 2 (50 mg/kg)
-Deep yellow in two males.
Group 3 (200 mg/kg)
-Deep yellow in two females.
-Light orange in all males and two females.
-Light brown in one female.
Group 4 (1000 mg/kg)
-Light orange in one female.
-Orange in two females.
-Red brown in three males and one female.
-Brown in two males and three females.
-Deep brown in five males and two females.

APPEARANCE
-Turbid urine in seven out of ten males and in two out of ten females of group 4 (1000 mg/kg).

OCCULT BLOOD
-Slightly higher degree of occult blood (score) in both sexes of groups 2 (50 mg/kg), 3 (200 mg/kg) and 4 (1000 mg/kg). In females this finding was found to be statistically significant (p < 0.05) for all treated groups.
At termination of the treatment-free recovery period these findings were, with the exception of a slightly higher incidence of occult blood in males of group 4.
reversible and comparable to those of the controls.
All other differences in the results of the urinalysis parameter were considered to be incidental or unrelated to the treatment, and of normal biological variation for rats of this strain and age.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in organ weight and organ weight ratio were noted:
Mean kidney weight and kidney weight ratios were consistently higher in all treated groups when compared with the respective controls. The following statistically significant changes were observed when compared with group 1:
Absolute weight:
Female Group 3 (200 mg/kg) p < 0.01
Weight relative to body weight:
Female Group 3 (200 mg/kg) p < 0.01
Weight relative to brain weight:
Female Group 2 (50 mg/kg) p < 0.05
Female Group 3 (200 mg/kg) p < 0.01
The marginal to slight increase in kidney weight in treated females of group 2 and 3 after 4 weeks of treatment is considered to be an effect of treatment. For females of group 4 and all treated males consistently higher mean kidney weights and kidney weight ratios were observed, but the values did not reach statistical significance. The remaining statistically significant differences in group 4 (1000 mg/kg) after 6 weeks of the recovery period Included higher liver weight and liver to body weight ratio in males (p < 0.05), higher spleen weight in animals of both sexes (p < 0.05), higher spleen to body and brain weight ratios in females (p < 0.05) and lower testes to brain weight ratio in males. Since there were no other corresponding findings and no evidence for any dose-relationship, these findings are considered to be incidental and unrelated to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Greenish discoloration in several organs of group 2, 3 and 4 animals, including the kidneys, gastrointestinal tract, lymph nodes, urinary bladder and in a single cases the testes and lungs were deemed to be test item related. All other gross lesions were unremarkable and were within the range of common necropsy findings in Wistar rats of these ages.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Multifocal tubular degeneration was seen in group 4 rats - four males and all females (minimal to moderate in severity) at the end of treatment and two males and all females (minimal to slight in severity) following the recovery period.
This finding consisted of vacuolation and plasmolysis/karyolysis of the cortical tubular epithelium of all group 3 females and all group 4 animals. Although the pigment showed a positive reaction for lipofuscin in sections, stained by Nile Blue and PAS methods, it was thought more likely to be the test item or a metabolite thereof. A similar pigment (minimal to slight degree) was recorded in several lymph nodes of animals of all treated groups as well as stored in phagocytic mucosal cells in various intestinal segments of group 3 and 4 animals (main test and recovery). In these organs the pigment deposition was not associated with cellular degeneration.
Histopathologic examination showed an increase in incidence and grade of tubular degeneration in kidneys of treated rats when compared with control rats:

EXOGENOUS PIGMENT DEPOSITION
-Group 3 (200 mg/kg): incidence in 5 females, grade 1
-Group 4 (1000 mg/kg), both after 4 and 6 weeks: incidence in 5 males and 5 females, grade 1-2

TABULAR DEGENERATION
-Group 4 (1000 mg/kg), after 4 weeks: incidence in 4 males and 5 females, grade 1-2-3
-Group 4 (1000 mg/kg), after 6 weeks: incidence in 2 males and 5 males, grade 1-2

The other recorded microscopic findings did not vary significantly in incidence or severity between control and treated groups, nor between rats sacrificed at the end of the treatment period or following recovery phase. They were all spontaneous in nature and within the normal range of background morphologic alterations which may be recorded in rats of this strain and age.

Details on results:

The data recorded during the in-life phase of the study indicated no adverse changes or effects attributable to treatment with the test item. All animals were normal in appearance and behaviour and survived their assigned study periods.
The only treatment-related effect observed during the in-life phase of the study was an increased food consumption (absolute and relative) in male and female animals during the treatment period. However, as the differences at most occasions were not statistically significant and did not result in higher body weight, and as the relative food consumption during the recovery period was similar in high-treated animals and controls, the changes are considered to be of no toxicological significance.
The assessment of hematology and clinical biochernistry data indicated no remarkable findings between control and treated rats at termination of the treatment which are considered to be of toxicological significance. The hematological findings were restricted to animals of group 4 (1000 mg/kg) and induded lower hemoglobin concentration and hematocrit in females, higher total leucocyte counts and changes in differential leucocyte count in males. As all findings were found to be reversible following the 15-day recovery period and were within or close to the limits of the historical control data, the effects are considered not to be biologically significant.
The urine discoloration was assumed to be attributed to passive discoloration of the test item. The presence of occult blood in urine of all treated animals may be due to a masking effect of the test item or metabolites thereof with the reagent test-strip reaction. However, the few findings at urinalysis, i.e. urine discoloration, occult blood in urine and the marginal higher urine volume (statistically not significant) point to the kidney as a target organ.
At necropsy the increase in kidney weight and kidney weight ratios gave also some evidence for the kidneys to be a target organ of toxicity to the test item. This is in agreement with the histopathological findings which showed an increased incidence and grade of tubular degeneration accompanied by exogenous pigment deposition in kidneys of animals in group 3 and 4. Therefore, in this study the test substance is considered to have a dose-related nephrotoxic effect.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

ANALYTICAL RESULTS

Pretest

The mean concentrations of the homogeneity samples taken during acclimatization/pretest were found to be 116.5 %, 106.1 % and 103.9 % of the nominal concentrations for dose groups 2 (5 mg/ml), 3 (20 mg/ml) and 4 (100 mg/ml). respectively. The homogeneity varied in the range from -8 % to +15 % of the mean concentrations. The substance was found to be stable in bidistilled water at room temperature for at least two hours.

Test

The mean concentrations of the homogeneity samples taken during administration/treatment were found to be 118.2 %, 111.4 % and 100.7 % of the nominal concentrations for dose groups 2 (5 mg/ml), 3 (20 mg/ml) and 4 (100 mg/ml), respectively. The homogeneity varied in the range from -1 % to +2 % of the mean concentrations. The substance was found to be stable in bidistilled water at room temperature for at least two hours.

Applicant's summary and conclusion

Conclusions:

The NOAEL of the substance was established as 200 mg/kg body weight for male and female rats.

Executive summary:

The repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1981) and the method B.7 of the Directive 92/69/EEC. The substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle only (bidistilled water). Each dose-group comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then observed for further 15-day treatment-free period after which they were sacrificed.

Clinical signs, mortality, food consumption, body weights and ophtalmoscopic examinations were recorded during the treatment and recovery periods.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for haematology and biochemistry analyses. Urine samples were collected for urinalyses.

All animals were killed, necropsied and examined post mortem; histological examinations were performed on organs and tissues.

No clinical signs of adverse reaction to treatment were observed, except in one male animal of group at dose of 50 mg/kg which developed sedation, dyspnea and ruffled fur on test day 5 and was killed in extermis. All the other animals survived their assigned study periods. Body weight development was not affected by treatment. In dose-group of 1000 mg/kg, absolute and relative food consumption was increased compared to the other treatment groups and control. In all other groups the overall food consumption was in the same range for treated animals and controls. There were no treatment-related ophthalmoscopic findings.

The assessment of haematology and clinical biochemistry data Indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. Some statistically significant effects after 4 weeks of treatment but no longer statistically significant and comparable to those of the controls after the recovery period were recorded in the dose-group of 1000 mg/kg.

Urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. However, urine discoloration was noted in both sexes of dose-groups at 200 and 1000 mg/kg; a turbid appearance of some urine samples was observed in the highest dose-group. A slightly higher incidence of occult blood was noted in both sexes of all dose-group, but this latter finding may reflect a possible masking effect of the test item or metabolites thereof with the reagent test-strip-reaction. At termination of the recovery period these findings were, with the exception of a slightly higher incidence of occult blood in males of the highest dose-group, reversible and comparable to those of the controls. The urine volume of all treated animals was dose-related Increased.

Mean kidney weight and kidney weight ratios were consistently higher in all treated groups when compared with the respective controls. In the animals of all dose groups, a test-item related greenish discoloration was recorded in several organs, mainly kidneys, gastrointestinal tract and lymph nodes. Histopathological treatment-related findings were noted in the kidneys of the highest dose-group animals of both sacrifice points: multifocal tubular degeneration accompanied by an exogenous pigment deposition. Similar pigment was also seen in kidneys of dose-group of 200 mg/kg females, in lymph nodes of animals of all treated groups and in various intestinal segments, where it was not associated with adverse cellular reaction.  

In conclusion, the treatment with test item caused some effects in the kidneys at all dose levels which was indicated by higher kidney weights, some changes in urinalytical parameters and histopathological findings. Regarding the histopathological and urinanalytical findings a dose-relationship is evident. Based on these results the NOAEL of the substance was established as 200 mg/kg body weight for male and female rats.