Registration Dossier

Administrative data

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 1000 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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
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
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 6 weeks
- Weight at acclimatization: Males: 136.5-154.9 grams (mean 146.1 grams), Females: 116.2- 133.5 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: Pelleted standard Provimi Kliba 3433 (batch nos. 4/05 and 25/05) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland), ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 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
- Photoperiod (hrs dark / hrs light): 12/12
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 40821/A was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared weekly using a magnetic stirrer and stored at room temperature (20±5°C) in glass beakers. 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.
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 40821/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 859021) in which FAT 40821/A was administered by gavage to 2 rats per group and sex.
Observations and examinations performed and frequency:
- Mortality/viability: Observations for mortality/viability were recorded twice daily.
- Cage side observations: 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).
- Clinical observations: 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.
- Food consumption: The food consumption was recorded once during the acclimatization period and weekly thereafter.
- Body weight: Body weights were recorded weekly during the acclimatization, treatment and recovery and before necropsy.
- Functional observational battery: During week 4, relevant parameters 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 Activity monitor System. Animals were 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.
- Clinical laboratory investigations: 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 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, Heinz bodies, 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, Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, 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.
Sacrifice and pathology:
- 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 (incl. coagulating 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.
-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.
- Slides of organs and tissues that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. Because test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (kidneys, jejunum, ileum (peyer's patches), caecum, colon and mesenteric lymph nodes) 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:
- Viability/mortality: All animals survived until their scheduled necropsy.
- Cageside observations: No clinical observations of toxicological relevance were noted during daily observations. Presence of dark blue-colored feces in both male and female rats treated with 200 mg/kg/day (from day 9 of treatment until necropsy) and 1000 mg/kg/day (from day 2 of treatment until day 5 of recovery) was observed. Reversibility of the coloration was noted during recovery. This passive finding was considered to be related to the test item and is commonly noted after oral administration of dyestuffs. One female treated with 1000 mg/kg/day presented temporary rales (on treatment day 17).
- Clinical observations: Rales were noted in one female treated with 1000 mg/kg/day during treatment week 3. This temporary finding was considered to be of no toxicological relevance. The other animals presented no clinical signs during treatment weeks 1-3.
- Functional observational battery: Grip strength: There were a number of findings that distinguished test item-treated animals from controls. However, all findings were considered as incidental. Male rats treated with 50 mg/kg/day or with 200 mg/kg/day had increased mean forelimb grip strength (both p<0.01) when compared to control animals but no dose response relationship could be established. In female rats treated with 200 mg/kg/day, decreased mean hind limb grip strength was recorded (p<0.05). Locomotor Activity: The total locomotor activity of test item-treated rats of both sexes was not significantly affected at any dose level. Males treated with 50 mg/kg/day had a lower locomotor activity during the 30-40 minutes measurement interval (p<0.05) when compared to control males. As no dose response relationship could be established and as this finding was observed only in the individual gender, it was considered to be fortuitous.
- 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 the treatment and recovery periods.
- Body weights: At treatment day 22, female rats treated with 1000 mg/kg/day presented lower mean body weights than the control females (p<0.05) but this transient finding was considered to be of no toxicological relevance; these rats were already marginally smaller on day 1 of treatment (approx. -5%) and the body weights compared favorably on day 28 of treatment. No test item-related changes of toxicological relevance were noted during the treatment period in male and female mean body weight gain, at any dose level. Mean body weights and mean body weight gain of the recovery rats of both sexes were comparable with that of their respective controls.
- Clinical laboratory investigations: Hematology: No test item-related changes were noted after treatment in rats of either sex at 50 mg/kg/day or at 200 mg/kg/day. At treatment end the following test item-related findings were observed: Male rats treated with 1000 mg/kg/day presented a decreased relative low fluorescence reticulocyte maturity index (L-Reti, p<0.05) and increased high fluorescence reticulocyte maturity index (H-Reti, p<0.05), which values remained within the range of the historical control data. Decreased values of red blood cells (p<0.05), hemoglobin (p<0.01), hematocrit (p<0.01) and relative eosinophils (p<0.05) were noted in females treated with 1000 mg/kg/day. All the values remained within the range of the historical control data. Significantly decreased absolute eosinophils concentrations were recorded in females treated with 1000 mg/kg/day (p<0.01), which were below the range of the historical control data. All these findings are considered to be indicative of a compensated anemia in males and slight anemia in females treated with 1000 mg/kg/day. As these findings reverted during the treatment-free recovery period, these changes were considered to be test item related but not adverse. Increased methemoglobin (MetHb) concentrations were recorded in males treated with 200 mg/kg/day (p<0.05) and in rats of both sexes treated with 1000 mg/kg/day (p<0.01 in males, p<0.05 in females). However, no Heinz bodies were seen in test item-treated males and females at any dose level. Although the methemoglobin concentrations measured in test item-treated males were slightly above the range of the historical control data, the values recorded in control males also reached the maximal values of the historical control data. Therefore, these findings were considered to be test item unrelated. MetHb values recorded in test item-treated females (at all dose levels) remained within the range of the historical control data. After the recovery period, significant decreases in hemoglobin concentration distribution width (HDW, p<0.01 in males), activated partial thromboplastin time (PTT, p<0.05 in males), relative neutrophils (p<0.05 in females) and increased relative monocytes (p<0.05 in males) and relative basophils (p<0.05 in females) in rats previously treated with 1000 mg/kg/day were noted. As all these findings remained within the range of the historical control data and appeared during the treatment-free recovery period, they were considered to be of no toxicological relevance.
- Clinical laboratory investigations: Clinical biochemistry: At treatment end the following test item-related findings were observed: A dose related decrease in the creatinine concentrations was noted in rats of both sexes, which was only significant in females at 200 mg/kg/day (p<0.01, values within the range of the historical control data) and in males and females at 1000 mg/kg/day (both p<0.01, which values were below the range of the historical control data). These findings were considered to be adaptive as corroborated by renal tubular hypertrophy. After recovery, the creatinine concentrations in previously test item-treated males were lower than that in control males (p<0.01) but remained within the range of the historical control data. Male and female rats treated with 1000 mg/kg/day had significantly increased total bilirubin concentrations (both p<0.01), which values exceeded the ranges of the historical control data. These findings were considered to be test item related but non adverse and may be possibly due to hematology changes. In male and female rats treated with 1000 mg/kg/day, increased sodium concentrations were measured (both p<0.05), which values remained within the range of the historical control data. These findings may be considered as adaptive as corroborated by renal tubular hypertrophy. Although the alanine aminotransferase (ALAT) activity was reduced in males treated with 200 mg/kg/day (p<0.05) and in females treated with 1000 mg/kg/day (p<0.01), the values remained within the range of the historical control data. No toxicological relevance is associated with this change. Increased (at 200 mg/kg/day, p<0.05), respectively decreased (at 1000 mg/kg/day, p<0.01) inorganic phosphorus (P04-in) concentrations were noted in female rats. These parameters remained within the range of the historical control data and were considered toxicologically irrelevant. In female rats treated with 1000 mg/kg/day, decreased globulin (p<0.01) concentrations were noted, which values remained within the range of the historical control data. Statistically significant decreased urea (p<0.05) concentrations were also measured in females at 1000 mg/kg/day, which values were below the range of the historical control data. All these findings were considered to be of no toxicological relevance. All other differences of statistical significance noted in test item-treated rats of either sex were either dose-unrelated, or within the range of the historical control data or appeared during the recovery period and therefore were considered to be incidental.
- Clinical laboratory investigations: urinalysis: Yellow-brown to black urine (males) and yellow-green to deep-green urine (females) were observed in rats treated with 1000 mg/kg/day. These findings were considered to be test item related but not adverse, as the kidney was clearly an excretory pathway. Increased bilirubin concentrations were measured in rats of both sexes treated with 1000 mg/kg/day (both p<0.05), which values exceeded the range of the historical control data. These findings were considered to be an artifact of the urine discoloration. After four weeks of treatment, increased erythrocytes in males (p<0.05) and in females (p<0.01) treated with 1000 mg/kg/day were noted. Although the values exceeded the range of the historical control data in test item-treated females, these findings may be due to tubular integrity loss. After recovery, the increased leukocytes concentrations (p<0.05) observed in males previously treated with 1000 mg/kg/day were considered to be fortuitous.
- Organ weights: After 4 Weeks: At the end of the treatment period, the relative kidneys weight (kidneys-to-body weight ratio) was increased in male (p<0.05) and female (p<0.01) rats treated with 1000 mg/kg/day. These adaptive test item-related findings can be correlated with the kidneys lesions (tubular hypertrophy) observed microscopically. Male rats treated with 50 mg/kg/day presented increased an absolute adrenals weight and an increased adrenals-to-body weight ratio (both p<0.05). Female rats treated with 50 mg/kg/day had an elevated spleen-to-body weight ratio (p<0.05). Insofar as no microscopical changes were ascertained, these differences were considered as incidental. The heart-to-body weight ratio was increased in males treated with 1000 mg/kg/day (p<0.05) after four weeks of treatment. Insofar as no microscopical changes were ascertained, this difference was considered as incidental. After 6 Weeks: At the end of the recovery period, the kidneys weight of male and female rats previously treated with 1000 mg/kg/day returned to normal values. Decreased absolute and/or relative brain (p<0.05), liver (p<0.05), adrenals (p<0.01 for absolute, p<0.05 for relative) weights in females previously treated with 1000 mg/kg/day were probably due to slightly lower body weights and were within the range of the biological variation occurring in rats of this strain and age and therefore were without toxicological relevance.
- Macroscopic findings: At necropsy performed at the end of the treatment period, a bluish discoloration of the mucosa of the ileum (all rats), caecum (all rats) and colon (4 out of 5 males and all females) as well as a bluish discoloration of the mesenteric lymph nodes (all rats) was observed in all animals treated with 1000 mg/kg/day. The jejunum of 2/5 females treated with 1000 mg/kg/day also presented a bluish discoloration of the mucosa. At the end of the recovery period, the mucosa of jejunum (in 4 out of 5 males only) and ileum (in all males and in 4 out of 5 females) was black in animals previously treated with 1000 mg/kg/day. A black discoloration was also recorded in the mesenteric lymph nodes of all animals treated with 1000 mg/kg/day. These findings were related to the black color of the test item. The remaining few macroscopic findings such as pelvic dilatation of the kidneys, dark red foci in the seminal vesicles, watery cyst in one ovary, reddish foci or dark red foci in the thymus observed in few animals, and regularly distributed among control and treated groups, were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age in four-week studies and were considered incidental, reflecting the usual individual variability.
- Microscopic findings: At the end of the treatment period, test item-related microscopic lesions were observed in the kidneys of animals treated with 1000 mg/kg/day. These lesions consisted of a minimal cellular hypertrophy of the proximal cortical tubules that was reported as tubular hypertrophy. This finding correlated with the relative kidney weight increase. After two weeks of recovery, the kidneys of animals previously treated with 1000 mg/kg/day returned to normal. The remaining microscopic findings, regularly distributed among the groups, were within the range and severity of spontaneous background lesions, which may be observed in rats of this strain and age in this laboratory and considered to be of no toxicological significance.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
Critical effects observed:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
The NOAEL was determined to be 1000 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 toxicological relevance during daily and weekly observations (weeks 1-3) or during functional observational battery (week 4), no effects on grip strength, no effects on locomotor activity and no effects on food consumption and body weight. Non-adverse test item-related changes were noted in test item-treated rats at 1000 mg/kg/day and included: decreased relative low fluorescence reticulocyte maturity index and increased high fluorescence reticulocyte maturity index in males, decreased values of red blood cells, hemoglobin, hematocrit and eosinophils (absolute and relative) in females. These findings are considered to be indicative of a compensated anemia in males and a slight anemia in females, which reverted during the treatment-free recovery period. Also an increase in total bilirubin concentrations in males and females was observed. These findings may be possibly due to hematology changes. Test item-related changes of adaptive nature were found in the kidneys. Dose related decreases in the creatinine concentrations and increased sodium concentrations were noted in test item-treated rats of both sexes. These findings were correlated with renal tubular hypertrophy. At the end of the treatment period, relative kidney weight of males and females treated with 1000 mg/kg/day was increased. After two weeks of recovery, the kidney weights returned to normal values. The bluish or black discoloration of the mucosa of the ileum, caecum and colon as well as the bluish or black discoloration of the mesenteric lymph nodes recorded at necropsy in animals treated with 1000 mg/kg/day had no microscopic correlate. It is likely that this staining was due to the physical-chemical properties of the test item that, however, did not induce any morphological change to the organs involved. Microscopically, tubular hypertrophy of the kidneys was seen in animals treated with 1000 mg/kg/day that correlated with the relative weight increase of this organ. However in the absence of any sign of degeneration or inflammation associated to the hypertrophy, this change was considered as an adaptive rather than a toxic response of the kidneys. After two weeks of recovery the kidney changes (both the weight and the morphology) returned to normal. Based on the results of this study, 200 mg/kg body weight/day of the test substance was established as the no-observed-effect-level (NOEL) in both male and female animals. The no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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

Mode of Action Analysis / Human Relevance Framework

Additional information

A 5-day range finding study was performed to select the dosages for a 28-day oral toxicity study (RCC 2005). It was recommended to expose the animals to 0, 50, 200, or 1000 mg/kg bw of the test substance.

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 2005). 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 toxicological relevance during daily and weekly observations (weeks 1-3) or during functional observational battery (week 4), no effects on grip strength, no effects on locomotor activity and no effects on food consumption and body weight. Non-adverse test item-related changes were noted in test item-treated rats at 1000 mg/kg/day and included: decreased relative low fluorescence reticulocyte maturity index and increased high fluorescence reticulocyte maturity index in males, decreased values of red blood cells, hemoglobin, hematocrit and eosinophils (absolute and relative) in females. These findings are considered to be indicative of a compensated anemia in males and a slight anemia in females, which reverted during the treatment-free recovery period. Also an increase in total bilirubin concentrations in males and females was observed. These findings may be possibly due to hematology changes. Test item-related changes of adaptive nature were found in the kidneys. Dose related decreases in the creatinine concentrations and increased sodium concentrations were noted in test item-treated rats of both sexes. These findings were correlated with renal tubular hypertrophy. At the end of the treatment period, relative kidney weight of males and females treated with 1000 mg/kg/day was increased. After two weeks of recovery, the kidney weights returned to normal values. The bluish or black discoloration of the mucosa of the ileum, caecum and colon as well as the bluish or black discoloration of the mesenteric lymph nodes recorded at necropsy in animals treated with 1000 mg/kg/day had no microscopic correlate. It is likely that this staining was due to the physical-chemical properties of the test item that, however, did not induce any morphological change to the organs involved. Microscopically, tubular hypertrophy of the kidneys was seen in animals treated with 1000 mg/kg/day that correlated with the relative weight increase of this organ. However in the absence of any sign of degeneration or inflammation associated to the hypertrophy, this change was considered as an adaptive rather than a toxic response of the kidneys. After two weeks of recovery the kidney changes (both the weight and the morphology) returned to normal. Based on the results of this study, 200 mg/kg body weight/day of the test substance was established as the no-observed-effect-level (NOEL) in both male and female animals. The no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg body weight/day.

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.