reaction mass of: pentasodium bis[6-anilino-3,5'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III);tetrasodium [6-anilino-3,5'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato][6-anilino-5'-sulfamoyl-3-sulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III);trisodium bis[6-anilino-5'-sulfamoyl-3-sulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(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:

2003

Report date:

2003

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:

1995

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-290-0

EC Name:

-

Cas Number:

508202-43-5

Molecular formula:

Not applicable

IUPAC Name:

tricobalt(3+) dodecasodium tris(2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-6-(phenylamino)-3-sulfonatonaphthalen-1-olate) tris(2-[2-(2-oxido-5-sulfonatophenyl)diazen-1-yl]-6-(phenylamino)-3-sulfonatonaphthalen-1-olate)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanBrl

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
-Age at study initiation: 6 weeks
-Weight at study initiation:
♂: 127.1 -153.8 grams (mean 136.4 grams)
♀: 112.0 -130.4 grams (mean 120.6 grams)
-Housing: in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding.
-Diet: pelleted standard rat maintenance diet, ad libitum. The feed batch was analyzed for contaminants.
-Water: Community tap-water from Itingen, ad libitum in water bottles.
-Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
-Temperature: 17-23' °C
-Humidity: 30 - 70 %
-Air changes: 10-15 air changes per hour
-Photoperiod: 12 hours cycle dark/light
-Other: music duríng 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 balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
-Amount of vehicle: 10 ml/kg body weigh.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC method

Details on analytical verification of doses or concentrations:

SAMPLE PREPARATION AND STORAGE
Concentration, homogeneity and stabílity (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.
Test item/vehicle mixtures were prepared and about 2 g (weighed to the third decimal place) of each mixture was weighed into a 100-ml volumetric flask. These samples for analysis were stored deep-frozen (about -20 °C) until analysis.

-Reagents: purified water, Acetonitrile, Ammonium acetate.
-Standard Solutions: stock Solutions of the test item in purified water with concentrations in the range from 288.0 mg/ml to 329.1 µg/ml were prepared by weighing appropriate amounts of the test item accurately into 100-ml volumetric flasks. Then about 70 ml of purified water was added to each volumetric flask and dissolution was achieved using an ultrasonic bath. The flasks were filled to the mark with purified water. Finally, various standard solutions were prepared by respective dilution of these stock solutions with purified water to yield concentrations in the range from 5.760 µg/mL to 159.7 µg/ml. These standard solutions were used for calibration of the HPLC.
-Analysis of Samples: the samples were dissolved in about 70 ml of purified water using an ultrasonic bath. Then, the 100-ml volumetric flasks were filled to the mark with purified water. Depending on the dose group, the latter sample solutions were further diluted with purified water to yield concentrations within the calibration range. Finally, a defined aliquot was quantified by HPLC.
-High Performance Liquid Chromatographic Determination
Typical Apparatus pump: Merck-Hitachi L-6200A
Detector: Merck-Hitachi L-4200
Sampling unit: Pro Star Varian
Integration: EZ Chrom Software
Column: Discovery C-18; 5 µm; 150 x 4 mm
Eluent A: 3.85 g ammonium acetate in 1 L purified water
Eluent B: Acetonitrile
Gradient:
time min %A %B
0 90 10
20 60 40
25 60 40
26 90 10
40 90 10

Flow: 1.0 ml/min
Wave length: 559 nm
Injection volume: 100 µl

Injected samples were quantified by the peak areas' (counts) of the test item with reference to the calibration curve. The latter was obtained by correlation of the peak areas of the
standard solutions with their corresponding concentrations (µg/mL), using the following equation 1:
y = a + b * X
where
Y = Peak areas (sum of 4 peaks) of test item in injected sample [counts]
a = Y-axis intercept
b = Slope
X = Concentration of test item in injected sample [µg/ml]

The concentrations of test Item in vehicle were calculated according to equation 2:
C= (X * V * D* Q)/ (W * 1000)
where
C = Concentration of test item in vehicle [mg/ml]
X = Concentration of test item in injected sample calculated by equation 1 [µg/ml]
V = Final volume [ml]
D = Dilution factor
Q = Density of test item/vehicle mixtures [assumed to be 1.0 g/ml]
W = Weight of sample [about 2 g, weighed to the third decimal place]

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

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 0 and 1000 mg/kg bw: 10 males; 10 females per dose
- Dose groups 50 and 200 mg/kg bw : 5 males; 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

-Dose selection rationale: based upon the results of a non-GLP 5-day dose-range-finding study. The substance was administered by gavage to 2 rats per group and sex.

Examinations

Observations and examinations performed and frequency:

-MORTALITY / VIABILITY
-Time schedule: were recorded twice daily

DETAILED CLINICAL OBSERVATIONS
-Time schedule: The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28, and once daily during days 29-42 (recovery).

BODY WEIGHT
-Time schedule for examinations: Body weights were recorded weekly during pretest, treatment, recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the computer.

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

HAEMATOLOGY
-Time schedule for collection of blood: after 4 weeks
-Anaesthetic used for blood collection: Yes, isoflurane anesthesia.
-Animals fasted: The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
The following hematology parameters were measured:
Erythrocyte count
Reticulocyte maturity Index
Hemoglobin
Methemogtobin
Hematocrit
Total leukocyte count
Mean corpuscular volume
Differential leukocyte count
Red cell volume distribution width
Coagulation (thromboplastin time and activated partial thromboplastin time)
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Platelet (thrombocyte) count
Reticulocyte count

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were measured:
Glucose
Alkaline phosphatase
Urea
Gamma-glutamyl-transferase
Creatinine
Sodium
Bilirubin, total
Potassium
Cholesterol, total
Chloride
Triglycerides
Calcium
Phospholipids
Phosphorus inorganic
Aspartate aminotransferase
Protein, total
Alanine aminotransferase
Albumin
Lactate dehydrogenase Globulin
Glutamate dehydrogenase
Albumin Globulin ratio
Creatine kinase


URINALYSIS
-Time schedule: urine was collected dunng the 18-hour fasting period into a specimen vial.
The following urinalysis parameters were measured:
Volume (18 hours)
Specific gravity (relative density)
Colour
Appearance
PH
Protein
Glucose
Ketone
Urobilinogen
Bilirubin
Blood

OTHER:
FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. Detailed Clinical Observations were performed weekly under week 4.
GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.
LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were randomized and monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

Sacrifice and pathology:

GROSS PATHOLOGY
AII animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated):
Adrenal glands
Aorta
Bone (stemum, femur including joint)
Bone marrow (femur)
Brain (cerebrum, cerebellum, joint)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes with optic nerve (fixed in Davidson’s solution)
Harderian gland (fixed in Davldson's solution)
Heart
lleum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland (exorbital)
Liver
Lungs (infused with formalin at necropsy)
Lymph nodes (mesenteric, mandibular)
Mammary gland area
Nasal cavity
Ovaries
Pancreas
Pituitary gland
Prostate gland
Rectum
Salivary glands (mandibular, sublingual)
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord (cervical, midthoracic, lumbar)
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland)
Tongue
Trachea
Urinary bladder (Infused with formalin at necropsy)
Uterus
Vagina
Gross lesions

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:
Ovaries
Brain
Heart
Liver
Thymus
Kidneys
Adrenals
Spleen
Testes
Epididymides
The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOPATHOLOGY
AII organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 micrometres, and stained with haematoxylin and eosin.
Slides of all organs and tissues listed in boldface type which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist.
As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (spleen and bone marrow) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyse the grip strength, locomotor activity, body weight, organ weights and ratios:
•The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
•The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
•Student's t-test was applied to grip strength and locomotor activity.
•Fisher’s exact-test was applied to the macroscopic findings.
For clinical laboratory data, quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to Bartlett.
Altenatively, if the variances were considered to be heterogeneous (p^0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were then compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p ≤ 0.05).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related clinical signs of toxicological relevance noted during daily observations.
Dark-red feces, dose-related in severity, was noted soon after the beginning of treatment in rats treated with 200 mg/kg/day (day 3) and in rats treated with 1000 mg/kg/day (day 2). This finding is commonly seen in oral gavage studies with dyestuffs and is considered to be a passive effect. Soft feces was noted on day 13 of treatment in males treated with 1000 mg/kg/day. This isolated incident was considered to be incidental. One female treated with 1000 mg/kg/day had an ocular abnormality in the structure of the right pupil durnig weeks 1-3. As this finding was already noted during pretest, it was considered to be a congenital finding and therefore of no relevance.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes of toxicological significance were noted at any dose level in the mean body weights or mean body weight gain.
At 1000 mg/kg/day, the mean body weights and mean body weight gain of males were marginally lower from day 15 and day 22 of treatment, respectively. These differences did not attain statistical significance, nor were such differences seen in the females at this dose level. These minor differences were considered to be unrelated to the test item. The mean body weights of the females were unaffected at all dose levels tested when compared with those of the control females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test item-treated animals was unaffected during the treatment and recovery phases of the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks' treatment, significantly increased (p<0.01) red blood cell counts were noted in males and females treated with 1000 mg/kg/day, as well as increased hemoglobin (p<0.01), hematocrit (p<0.01) and red cell distribution width (p<0.01). The mean haemoglobin distribution width was also increased in the males (p<0.05) and females (p<0.01) when compared with the respective controls. After 2 weeks' recovery, significantly increased red blood cell counts persisted in males (p<0.05) and females (p<0.01) treated previously with 1000 mg/kg/day, whereas increased haemoglobin (p<0.05) and haematocrit (p<0.01) were seen only in females.
Significantly higher mean absolute and relative reticulocyte counts were noted in both sexes (p<0.01) after 4 weeks' treatment with 1000 mg/kg/day; this finding was also seen to a lesser extent in the males treated with 200 mg/kg/day, when compared with the controls.
Significantly increased (p<0.01) absolute and relative reticulocyte counts persisted in females treated previously with 1000 mg/kg/day.
The reticulocyte maturity Indices of rats treated with 1000 mg/kg/day revealed a "left-shift" towards high fluorescence reticulocytes {p<0.01), accompanying by statistically significant reductions in low fluorescence (p<0.01) and middies fluorescence (p<0.05 in males, p<0.01 in females) reticulocytes when compared with the respective control values.
Many of the aforementioned parameters exceeded the limits of the historical control data and were therefore considered to be test item-related changes, and possibly indicative of a compensated reticulocytisis.
The mean relative eosinophil counts were significantly reduced in males {p<0.05) and females (p<0.01) treated with 1000 mg/kg/day and in females {p<0.05) treated with 200 mg/kg/day when compared with the controls, whereas the mean absolute eosinophil counts of females treated with 200 mg/kg/day (p<0.01) and 1000 mg/kg/day (p<0.01) when compared with the controls. These findings reverted to control levels after the recovery period. After the 2-week recovery period, the mean neutrophil count of the males treated previously with 1000 mg/kg/day was significantly elevated (p<0.05) when compared with the controls. All differences remained within the ranges of the historical control values and were therefore considered to be without toxicological relevance.
When compared with the controls, the mean platelet count was significantly reduced in males (p<0.01) treated with 1000 mg/kg/day after 4 weeks' treatment and 2 weeks' recovery. The activated partial thromboplastin time was significantly prolonged (p<0.01) in females treated with 1000 mg/kg/day, although this difference remained within the range of the historical control values.
All other values noted after 4 weeks' treatment and 2 weeks' recovery compared favorably with those of the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Reduced glucose levels (p<0.01) were noted after 4 weeks in males and females treated with 1000 mg/kg/day when compared with controls, whereas reduced urea levels (p<0.05) and increased total bilirubin (p<0.05) were noted after 4 weeks in males treated with 1000 mg/kg/day. Significantly increased (p<0.01) activity of aspartate aminotransferase was noted after 4 weeks in males treated with 1000 mg/kg/day, and significantly reduced activity of alanine aminotransferase was noted after 4 weeks' treatment in males (p<0.05) and females (p<0.01), and after 2 weeks' recovery in females (p<0.05). Lactate dehydrogenase was increased in males (p<0.05) and females (p<0.05) following 4 weeks of treatment with 1000 mg/kg/day. These changes occasionally exceeded the ranges ot the historical control values, and were considered to indicate that liver was the location of the metabolism of the test item.
Glutamate dehydrogenase was increased in males treated with 200 mg/kg/day and 1000 mg/kg/day when compared with the control males after 4 weeks, whereas females were unaffected.
All remaining changes noted following the 4-week treatment period (decreased potassium in males at 1000 mg/kg/day/increased potassium in females at 1000 mg/kg/day, increased chloride in males at 200 mg/kg/day and 1000 mg/kg/day, decreased calcium in males at 50 mg/kg/day and 1000 mg/kg/day (also seen in the latter group after 2 weeks of recovery), decreased protein in males treated with 50 mg/kg/day and 1000 mg/kg/day after 4 weeks of treatment (as well as In females treated with 1000 mg/kg/day after 2 weeks recovery), and decreased albumin in males treated with 50 mg/kg/day after 4 weeks of treatment) either remained within the ranges of the historical control data, were only observed in one sex, or resulted from a divergent control value and were considered to be incidental changes of no toxicological relevance.
Decreased creatinine (p<0.05) and triglycerides (p<0.01) were noted after 2 weeks' recovery in females treated previously with 1000 mg/kg/day, although the latter difference was considered to be due to a high control value.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinary pH was increased in males (p<0.01) and females (p<0.05) treated with 1000 mg/kg/day, when compared with the controls. Although only the value for the males exceeded the historical control data, this finding was considered likely to be test item-related. The protein levels noted in the males after 4 weeks were significantly increased (p<0.01), but this was considered to be due to a low control value. Bilirubin levels were increased after 4 weeks in males (p<0.01) and females (p<0.01) treated with 1000 mg/kg/day when compared with controls. Although a marginal increase was also noted in both sexes treated with 200 mg/kg/day, all differences remained within the ranges of the historical control data.
The elevated erythrocyte levels seen in males treated with 200 mg/kg/day and 1000 mg/kg/day also remained within the ranges of the historical control data, although the differences seen in the latter group persisted after the 2-week recovery period. Leukocyte counts were increased in males (p<0.01) and females (p<0.05) treated with 1000 mg/kg/day when compared with the controls, but this finding was reversible after the 2-week recovery period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 Weeks significantly elevated absolute spleen weights were noted in females treated with 200 mg/kg/day (p<0.05) and significantly elevated spleen-to-body weight ratios were noted in females treated with 1000 mg/kg/day {p<0.05) when compared to the controls. These differences were considered to be test item-related. The remaining absolute and relative organ weights noted in the test item-treated females were considered to be similar to those of the controls. Significantly reduced absolute liver weights (p<0.01) and absolute thymus weights (p<0.05) were noted in males treated with 1000 mg/kg/day when compared with the controls. The significantly lower absolute brain weight (p<0.05) noted in males treated with 1000 mg/kg/day was considered to result from the lower body weights of these males; the brain-to-body weight ratio compared favorably with that of the control males. These differences were, in the absence of microscopic changes, considered to be incidental. The significantly elevated heart-to-brain weight ratios (p<0.05) noted in males treated with 50 mg/kg/day and the elevated heart-to-brain and heart-to-body weight ratios (p<0.05) noted in males treated with 200 mg/kg/day were not dose-dependent and therefore also considered to be incidental.
After 6 Weeks the absolute and relative organ weights of rats previously treated with the test item compared favorably with those of the respective controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes occurred in males and females at 1000 mg/kg/day. At the end of the treatment period, one of five males had violet discoloration of the stomach and ileum, four of five males of the mesenteric lymph node, and five of five males had such discoloration of the large intestino and dark red discoloration of the kidneys. Two of five females had violet discoloration of the mesenteric lymph node, and five of five females had violet discoloration of the large intestine, as well as dark red discoloration of the kidneys. Following the recovery period, four of five males and three of five females had black discoloration of the kidneys. Other necropsy findings were considered incidental owing to their nature and low incidence.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Bone marrow
At the end of the treatment period, fatty atrophy of the bone marrow had decreased incidence and average grading in males and females at 1000 mg/kg/day. Following the recovery period, only low grade fatty atrophy occurred in the bone marrow of a few control animals. This finding was considered to be of no toxicological significance.
Kidneys
At the end of the treatment period, no difference between the treated and control male and female groups was observed.
Following the recovery period, two kinds of changes occurred in the proximal renal tubules of the mates at 1000 mg/kg/day: an increased average grading of tubular hylaine change and occurrence of minimal to marked cytoplasmic inclusions occurred as well. The tubular hyaline change in the males had features compatible with deposition of alpha-2-macroglobulins. The cytoplasmic inclusions in both sexes were aggregates of granular material located in intracytoplasmic vacuoles.
Spleen
At the end of the treatment period, splenic extramedullary hematopoietic activity had increased average grading in males at 200 mg/kg/day and in both sexes at 1000 mg/kg/day.
Following the recovery period, this finding had still higher average grading in males at 1000 mg/kg/day.
Incidental findings The type, incidence, distribution among the groups and severity of all other findings did not indicate effects related to the treatment with the test Item. The observed findings corresponded to those naturally occurring in the untreated control rats of this colony, age, and type of study.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY
There were no test item-related clinical signs of toxicological relevance noted during functional observational battery (week 4).
One female treated with 1000 mg/kg/day had an ocular abnormality in the structure of the right pupil during the functional observational battery at week 4. As this finding was already noted during pretest, It was considered to be a congenital finding and therefore of no relevance.

GRIP STRENGTH
No test item-related changes were noted in the fore- or hindlimb grip strength of males or females at any dose level.
In females treated with 50 mg/kg/day, a statistically significant increase (p<0.05) in the forelimb grip strength was noted, whereas females treated with 200 mg/kg/day had significantly increased (p<0.05) hindlimb grip strength when compared with the controls. These contrary differences were considered to be incidental.
The fore- and hindlimb grip strength values of the males and females treated with 1000 mg/kg/day were similar to those of the respective controls.

LOCOMOTOR ACTIVITY
The mean locomotor activity of the test item-treated males and females were considered to be unaffected when compared to the respective control values.
In males treated with 200 mg/kg/day, the locomotor activity was significantly increased (p<0.01) during 30-45 minutes and the total locomotor activity (0-60 minutes) was significantly higher (p<0.05) than that of the respective control value. These differences were considered to be Incidental as the locomotor activity of the males treated with 1000 mg/kg/day compared favorably with those of the control males. In females treated with 50 mg/kg/day, the locomotor activity was significantly reduced (p<0.01) during 30-45 minutes when compared with the control value. Since females treated with 200 mg/kg/day were unaffected, the differences seen at 50 mg/kg/day were considered to be fortuitous.

Effect levels

1

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

organ weights and organ / body weight ratios

urinalysis

2

Dose descriptor:

NOEL

Effect level:

50 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

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

ANALYTICAL RESULTS

I) The mean concentratlons of the homogeneity samples were found to be 101.0 %, 105.8 %, and 101.3 % of the nominal concentrations of dose group 2 (5 mg/ml), dose group 3 (20 mg/ml), and dose group 4 (100 mg/ml), respectlvely. The individual concentrations varied in the range from -4% to +3% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle. The test item was stable in the vehicle under storage conditions for 7 days.

II) The mean concentrations of the homogeneity samples were found to be 104.3 %, 106.0 %, and 98.6 % of tha nominal concentrations of dose group 2 (5 mg/ml), dose group 3 (20 mg/ml), and dose group 4 (100 mg/ml), respectively. The individual concentrations varied in the range from -1 % to +1 % of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Applicant's summary and conclusion

Conclusions:

The NOAEL (oral, 28 d) for repeated dose toxicity was established as 200 mg/kg b.w./day.

Executive summary:

The repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1995) and the method B.7 of the Directive 96/54/EC. 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, bidistilled water, only. The groups 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 allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for haematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, the spleen and bone marrow were examined to establish a no-effect level.

No mortality, no clinical signs of toxicological relevance during daily and weekly observations, no effects on functional observational battery (including locomotor activity and grip strength), and no effects on food consumption or body weight. Test item-related findings were generally restricted to changes in haematology parameters (indicative of compensated reticulocytosis) at 1000 mg/kg/day, changes in clinical biochemistry (indicative of changes in liver metabolism) at 1000 mg/kg/day, changes in urinalysis parameters (indicative of the renal pathway for elimination) at 1000 mg/kg/day, elevated spleen weights (females only) at 1000 mg/kg/day, macroscopic changes (mostly discoloration) in the digestive tract, kidney and mesenteric lymph nodes at 1000 mg/kg/day, microscopical findings in the bone marrow (fatty atrophy) at 1000 mg/kg/day, spleen (increased extramedullary hematopoietic activity) at 200 mg/kg/day (males only) and 1000 mg/kg/day (both sexes), and kidneys (tubular hyaline change in males and cytoplasmic inclusions in both sexes) at 1000 mg/kg/day.

Based on the results of this study, 50 mg/kg b.w./day of the test item was established as NOEL. Insofar as the changes seen at 200 mg/kg/day were noted in males only and reversible, 200 mg/kg b.w./day is considered to be NOAEL.