Sodium 5-({4-chloro-6-[ethyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-(1-sulfonato-2-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Endpoint summary

Description of key information

Based on the results obtained in the 28 day repeated toxicity study by oral gavage, the NOAEL was determined to be 200 mg/kg bw for male and female.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 6 weeks
- Weight at acclimatization: Males: 141.9-156.3 grams (mean 150.0 grams), Females: 119.0- 130.0 grams (mean 123.5 grams)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 (batch nos. 34/04, 42/04 and 53/04) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland), ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days, under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
Values outside of these ranges occasionally occurred. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC.
- Photoperiod (hrs dark / hrs light): 12/12 with music during the light period

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bidistilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. FAT 40'817/A 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 (20±5°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Dose volume: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (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. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
50, 200, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

30 males and 30 females;
Groups 0 mg/kg/day and 1000 mg/kg/day: 10 males; 10 females
Groups 50 mg/kg/day and 200 mg/kg/day: 5 males; 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

In this subacute toxicity study, FAT 40'817/A 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 that 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.

Rationale for dose level selection: Based upon the results of a non-GLP 5-day doserange-finding study (RCC Study Number 855610) in which FAT 40'817/A was administered by gavage to 2 rats per group and sex. Animals treated with 1000 mg/kg/day showed a passive test item-related effect (dark feces).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
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).
Observations for mortality/viability were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

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

FOOD CONSUMPTION AND COMPOUND INTAKE :
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 RCC computer.

HAEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS: Yes
Blood and urine sampling: after 4 weeks and 6 weeks. Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. 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 microhematocrit glass capillary tube. Urine was collected during the 18-hour fasting period into a specimen vial. The assays were performed at RCC Ltd (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.

The following hematology parameters were determined:
Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Total leukocyte count, Differential leukocyte count, Coagulation:, Thromboplastin time, Activated partial thromboplastin time.

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

The following urinalysis parameters were determined:
Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery: During week 4, relevant parameters (appearance, motor, behavior, respiration, reflexes, miscellaneous) from a modified Irwin screen test were evaluated in all animals.
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 AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH. 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 10-minute intervals as well as the total activity of the measuring period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Sacrifice:
after 4 weeks and after 6 weeks. All 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 (sternum, femur including joint), Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum 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, mid-thoracic, 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: Brain, Heart, Liver, Thymus, Kidneys, Adrenals, Spleen, Testes, Epididymides, Ovaries.
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: Yes
The following organs (Adrenal glands, Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Ovaries, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions) were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin. Additionally slides of all animals (groups 1-4) were stained with Papanicolaou (PAP) for better identification and quantification of the test item, a dye with intrinsic red color.

Slides of all organs and tissues listed above, under histopathology, that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. Because treatment-related morphologic changes were detected in the kidneys of high-dose animals, the same organs (kidneys) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were 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.
• Fisher's exact-test were applied to the macroscopic findings.

The following statistical methods were used for statistical analysis of clinical laboratory data:
• Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p≤0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were 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).
• Ordinal data such as urine sediment were analyzed using the Kruskal-Wallis test. If this test was significant (p≤0.05), comparisons were made between the
control group and each of the treatment groups using Dunn's test.

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy.
All animals were without clinical signs during weeks 1-3.

GENERAL CAGESIDE OBSERVATIONS
Dark red feces were noted both in males and females treated with 1000 mg/kg/day. These test item-induced passive findings reversed two days after the end of treatment (recovery phase).
No clinical observations of toxicological relevance were noted during daily observations.

BODY WEIGHT AND WEIGHT GAIN
No test item-related changes of toxicological relevance were noted in male and female mean body weights at any dose level. Mean body weight gain was decreased in males treated with 200 mg/kg/day at day 15 (p<0.05) and at the end of the study (day 28, p<0.05) when compared to the control rats. Mean body weight gain of test item-treated females was comparable to that of control females at all dose levels. After recovery, no statistically significant difference between mean body weight gain of controls and test item-treated male and female rats was observed.

FOOD CONSUMPTION
The mean daily food consumption and the relative food consumption of the test item-treated males and females compared favorably with their respective controls during treatment and recovery periods.

HAEMATOLOGY
No test item-related changes of toxicological relevance were noted in the hematology parameters at any dose level. The following differences were noted in the hematology parameters after four weeks' treatment and either remained within the range of the historical control data, were without clear dose response relationships, or were unsupported by concomitant changes in related parameters:
• increased mean relative low fluorescence reticulocyte count (p<0.05) in males treated with 50 mg/kg/day;
• decreased mean relative high fluorescence reticulocyte count in males treated with 50 mg/kg/day and 1000 mg/kg/day (both p<0.05);
• decreased mean total leukocyte count (p<0.05) in males treated with 200 mg/kg/day;
• increased neutrophils concentration in males treated with 200 mg/kg/day (P<0.05);
• decreased absolute lymphocyte concentration in males receiving 50 mg/kg/day (p<0.05) and 200 mg/kg/day (p<0.01). the mean absolute lymphocyte concentration (G/l) in males treated with 200 mg/kg/day was only marginally lower than the range of the historical data.
• decreased mean relative lymphocyte count (p<0.05) after four weeks in males treated with 200 mg/kg/day; and increased relative and absolute lymphocyte concentrations (p<0.01 and p<0.05 respectively) in females treated with 1000 mg/kg/day;
• increased activated partial thromboplastin time in males receiving 200 mg/kg/day (p<0.05) and decreased activated partial thromboplastin time (p<0.05) in females receiving 50 mg/kg/day and increased prothrombin time (p<0.01) in females treated with 1000 mg/kg/day;
• increased mean total leukocyte counts in females treated with 50 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.05);
• increased mean absolute neutrophil concentration (p<0.05) in females treated with 50 mg/kg/day;
• decreased mean relative neutrophils concentration in females receiving 1000 mg/kg/day (p<0.05).
After recovery, the erythrocyte count (p<0.01), hemoglobin and hematocrit concentrations were increased (both p<0.05) in males initially treated with 1000 mg/kg/day and an increased platelet count (p<0.01) was noted in females initially treated with 1000 mg/kg/day; but remained within the historical control data.

CLINICAL CHEMISTRY
No test item-related differences of toxicological relevance were noted in the clinical biochemistry parameters after four weeks' treatment at any dose level in both males and females. The following differences were noted in the hematology parameters after four weeks' treatment and either remained within the range of the historical control data, were without clear dose response relationships, or were unsupported by concomitant changes in related parameters:
• increased sodium concentration (p<0.01) and decreased inorganic phosphorus (p<0.05) in males treated with 50 mg/kg/day;
• increased albumin concentration in males treated with 200 mg/kg/day (p<0.05);
• increased potassium (p<0.01) concentrations in males receiving 1000 mg/kg/day;
• increased total bilirubin in males and females receiving 1000 mg/kg/day (p<0.01);
• females treated with 1000 mg/kg/day showed increased triglyceride concentrations (p<0.01) and decreased urea concentration (p<0.05).
After recovery, males treated with 1000 mg/kg/day showed an increased creatinine concentration (p<0.05) and a decreased triglyceride concentration (p<0.05). Both control and test item-treated animals showed lower triglyceride concentrations than the range of the historical control data.

URINALYSIS
No test item-related differences of toxicological relevance were noted in the urinalysis parameters at any dose level in both males and females. The following differences either remained within the range of the historical control data or were without a clear dose-response relationship could be established.
• increased urine volume at 200 mg/kg/day (p<0.05) and at 1000 mg/kg/day (p<0.01)
• increased urine pH value in males treated with 200 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.01);
• decreased relative density of the urine (p<0.05) in males receiving 200 mg/kg/day;
• yellow/brown to red color of the urine in 9/10 males and 6/10 females treated with 1000 mg/kg/day which returned to normal coloration during recovery;
• increased pH value (p<0.01), nitrite- (p<0.01), ketone- (p<0.05) and leukocyte (p<0.05) concentrations in females treated with 1000 mg/kg/day.
Bilirubin concentration in urine of males and females treated with 1000 mg/kg/day exceeded largely the historical control data but these findings were due to the high concentration of dyestuff given and were therefore considered as artifacts.

NEUROBEHAVIOUR
Breathing noise (grade 1) during respiration was observed during week 4 for one female treated with 1000 mg/kg/day. All other animals were without clinical signs during week 4.
No test item-related changes were noted in mean fore- and hind limb grip strength of males and females at any dose level.
Males treated with 1000 mg/kg/day presented a statistically significant higher total locomotor activity (+33.7%) compared to the control animals (p<0.05). At 1000 mg/kg/day, elevated locomotor activity was seen from 0-10 minutes in males (p<0.05) and females (p<0.05) when compared with the respective controls. Although these initial changes were considered to be test item related, later differences seen in males (50-60 minutes, p<0.05) and females (10-20 minutes, p<0.05) were seen only in the individual genders and therefore considered to be incidental. Females treated with 50 mg/kg/day showed a strong decrease in their locomotor activity during the 30-40 minutes measurement interval (p<0.05), but these differences were not seen at higher doses and therefore considered to be incidental.

ORGAN WEIGHTS
After 4 Weeks
In males treated with 50 mg/kg/day, a significant decrease in mean absolute and relative spleen weight (both p<0.05) was observed after four weeks' treatment. Insofar as no dose response relationship was evident, these differences were considered as incidental. No difference in mean absolute or relative organ weights was noted after four weeks' treatment in females at any dose level.
After 6 Weeks
No difference in mean absolute or relative organ weights was noted after the recovery period both in males and females at any dose level.

GROSS PATHOLOGY
At the end of the treatment and following recovery period, a bilateral (reddish or dark red) discoloration of the kidneys was recorded in all main study and recovery animals treated with 1000 mg/kg/day. This change was assessed as a test item-related gross lesion.
All other macroscopic findings consisting of discolored foci at the thymus (1/10 males vehicle-treated, 1/5 females receiving 50 mg/kg/day and 1/10 males treated with 1000 mg/kg/day) were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age in oral toxicity studies and were considered incidental, reflecting the usual individual variability.

HISTOPATHOLOGY:
There were a number of findings that distinguished test item-treated animals from controls.
Kidneys:
• minimal to moderate vacuolar tubulus cell degeneration in all test item-treated animals receiving 1000 mg/kg/day (after four weeks' treatment and recovery). The vacuoles contained minimal to moderate amounts of red granular pigment. Due to its intrinsic color, the pigment was better identifiable and quantifiable in slides stained with Papanicolaou (pale red);
• increased incidence and mean grade of tubular basophilia in animals treated with 1000 mg/kg/day after recovery. Some of the basophilic tubular cells showed vacuolar degeneration of the cytoplasm with red granular pigment in the cytoplasmic vacuoles. Due to its intrinsic color, the pigment was better identifiable in slides stained with Papanicolaou (pale red).
Incidental microscopic findings:
Additionally, a variety of other changes were found in this study. They commonly occur in laboratory rats of this strain and age under the conditions of oral toxicity studies. Neither their incidences nor their distribution or morphologic appearance gave any indication of a test item-related association.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The NOAEL was determined to be 200 mg/kg bw/day.

Executive summary:

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (50, 200, 1000 mg/kg bw) by repeated oral gavage for a period of 28 days. The study was comprised of 4 groups, the groups comprised 5 animals per sex that were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Oral administration of test substance for 28 days resulted in no mortality, no clinical signs of adverse nature during daily or weekly observations (weeks 1-3) or during functional observational battery (week 4), no effect on fore- and hind limb grip strength, no effect on locomotor activity, no effects on food consumption or body weight.

Test item-related findings were generally restricted to minor, reversible and non dose dependent changes in hematological, biochemistry and urine parameters. All these findings were considered to be non-adverse. Pathological examinations revealed that under the conditions of the study, the treatment with the test item induced test item-related changes in kidneys: A vacuolar tubulus cell degeneration was recorded in all test item-treated animals receiving 1000 mg/kg/day (after four weeks' treatment and recovery). Additionally, an increased incidence and mean grade of tubular basophilia was recorded after recovery in animals treated with 1000 mg/kg/day. The tubular basophilia was most likely a sign of an increased tubular regeneration after a preceding tubular damage, induced by the vacuolar degeneration of the tubulus cells. These renal changes were assessed as adverse effects reflecting the nephrotoxic properties of the test item at a dose of 1000 mg/kg/day. In this context it has to be considered that the increased incidence of tubular basophilia observed in animals treated with 1000 mg/kg/day only develop after a 14-day treatment-free recovery period. Animals treated with lowest doses of test item (50 mg/kg/day and 200 mg/kg/day) did not have a recovery period, but were necropsied already after 4 weeks. Therefore, some delayed changes recorded in animals treated with 1000 mg/kg/day probably may not be determined by this study design. As no adverse changes were recorded in animals receiving 50 mg/kg/day and 200 mg/kg/day, a no-observed-adverse-effect level (NOAEL) could be established at 200 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline study, klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity 28 days, oral:

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (50, 200, 1000 mg/kg bw) by repeated oral gavage for a period of 28 days (RCC 2004). The study was comprised of 4 groups, the groups comprised 5 animals per sex that were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Oral administration of test substance for 28 days resulted in no mortality, no clinical signs of adverse nature during daily or weekly observations (weeks 1-3) or during functional observational battery (week 4), no effect on fore- and hind limb grip strength, no effect on locomotor activity, no effects on food consumption or body weight. Test item-related findings were generally restricted to minor, reversible and non dose dependent changes in hematological, biochemistry and urine parameters. All these findings were considered to be non-adverse. Pathological examinations revealed that under the conditions of the study, the treatment with the test item induced test item-related changes in kidneys: A vacuolar tubulus cell degeneration was recorded in all test item-treated animals receiving 1000 mg/kg/day (after four weeks' treatment and recovery). Additionally, an increased incidence and mean grade of tubular basophilia was recorded after recovery in animals treated with 1000 mg/kg/day.The tubular basophilia was most likely a sign of an increased tubular regeneration after a preceding tubular damage, induced by the vacuolar degeneration of the tubulus cells. These renal changes were assessed as adverse effects reflecting the nephrotoxic properties of the test item at a dose of 1000 mg/kg/day. In this context it has to be considered that the increased incidence of tubular basophilia observed in animals treated with 1000 mg/kg/day only develop after a 14-day treatment-free recovery period. Animals treated with lowest doses of test item (50 mg/kg/day and 200 mg/kg/day) did not have a recovery period, but were necropsied already after 4 weeks. Therefore, some delayed changes recorded in animals treated with 1000 mg/kg/day probably may not be determined by this study design. As no adverse changes were recorded in animals receiving 50 mg/kg/day and 200 mg/kg/day, a no-observed-adverse-effect level (NOAEL) could be established at 200 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only oral repeated dose toxicity study available

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the test substance does meet the criteria of the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and therefore no classification is needed.