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Description of key information

In the available OECD 407 repeated toxicity study, the authors conclude that the NOAEL should be 50 mg/kg bw/day based on a slightly lower mean body weights in males treated with 200 mg/kg bw/day. However, the registrant is of the opinion that because the weight loss is only slight and the effect on body weight was reversible during the recovery period these effects are not adverse and the NOAEL should be 200 mg/kg bw.

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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:
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
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: 134.2-158.0 grams (mean 148.6 grams), Females: 111.0- 134.8 grams (mean 126.2 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 no. 17/03 and 40/03) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±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. 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. 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:
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, the test 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 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 849034) in which FAT 40'812/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 the first test item administration and once weekly (during 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 Acivity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). 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 15-minute intervals as well as the total activity of the measuring period.
- Clinical laboratory investigations: Blood samples for hematology and clinical chemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted 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. Urine was collected during the 18-hour fasting period into specimen vials. 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, Total leukocyte count, Differential leukocyte count, Methemoglobin, 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 (3 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 with coagulation glands, 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.
-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 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 the high-dose animals, those same organs from the mid- and low-dose group were examined.
Statistics:
The following statistical methods were used to analyze 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 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).
Details on results:
- Viability / Mortality: All animals survived until scheduled necropsy.
- General Cageside Observations (Daily): Dark red discoloration of the feces was noted in both sexes treated with 200 mg/kg/day from day 4 of treatment onwards and in both sexes treated with 1000 mg/kg/day from day 2 of treatment onwards. The severity of this finding was slight in the former group and slight to moderate in the latter group. This finding was considered to be a passive test item-related change, and is commonly seen following oral gavage of dyestuffs. Fecal discoloration continued for the first two days of the recovery period in both sexes treated previously with 1000 mg/kg/day. Localised alopecia (days 24-28 of treatment) and red ocular scabbing (days 24-26 of treatment) were noted in single females treated with 1000 mg/kg/day. These isolated findings were considered to be incidental changes and unrelated to the test item treatment.
- Detailed Clinical Observations (Weekly): Red ocular scabbing (week 3) was noted during weekly clinical observations in one female treated with 1000 mg/kg/day. This isolated finding was considered to be unrelated to the treatment with the test item.
- Functional Observational Battery: Red ocular scabbing (week 4) and localised hair loss were noted during the functional observational battery in single females treated with 1000 mg/kg/day. These isolated findings were considered to be unrelated to the treatment with the test item. The mean fore- and hindlimb grip strength of the test item-treated rats compared favorably with those of the control rats. With the exception of the males treated with 200 mg/kg/day (which had slightly elevated mean locomotor activity), the mean locomotor activity of the test item-treated males compared favorably with those of the control males. ln females, sporadic differences to the control values attained statistical significance (such as 30-45 minutes at 200 mg/kg/day and 1000 mg/kg/day (p<0.05), 0-15 minutes at 1000 mg/kg/day (p<0.05)). These differences were not considered to be related to the treatment with the test item. All other time points compared favorably with those of the control females.
- Food Consumption: The mean daily food consumption of the test item-treated rats was similar to those of the control rats during the treatment and recovery periods.
- Body Weights: The mean body weights and mean body weight gain values of male rats treated with 1000 mg/kg/day were slightly lower than those of the control males throughout the treatment period. The mean body weights of males treated with 200 mg/kg/day were significantly lower on day 22 (p<0.05) and day 28 (p<0.05) of treatment when compared with controls. In these rats, the slightly lower mean body weight gain noted on day 28 of treatment attained statistical significance (p<0.05) when compared with that of the control males. These differences were considered to be a possible effect of the test item treatment. In females, the mean body weights of the test item-treated rats were generally similar with those of the controls. Although the mean body weight gain of the females treated with 200 mg/kg/day were significantly lower on day 8 of treatment (p<0.05), this difference was dose-unrelated and transient. The body weight development during the recovery period was considered to be similar in all groups.
- Hematology: After 4 weeks' treatment, the mean leukocyte count was significantly lower (p<0.05) and the mean number of absolute lymphocytes were significantly increased (p<0.05) in males treated with 200 mg/kg/day when compared with the controls. In the absence of a dose-response relationship, these findings were considered to be incidental. The mean number of relative eosinophils was significantly increased (p<0.01) and the mean number of absolute monocytes were significantly reduced (p<0.01) in males treated with 1000 mg/kg/day when compared with the controls, but this difference remained within ranges of the historical control data and was therefore considered to be incidental. Marginally, but significantly higher methemoglobin levels were noted in females treated with 200 mg/kg/day (p<0.05) when compared with the controls, this was considered to be due to a low control value (the mean methemoglobin levels of all treated females were similar) and the difference remained within ranges of the historical control data. This finding was therefore considered to be incidental. After 2 weeks' recovery, the mean number of relative high fluorescence reticulocytes was significantly elevated in male rats previously treated with 1000 mg/kg/day (p<0.05), and the mean cell hemoglobin concentration was significantly increased in females treated previously with 1000 mg/kg/day, but in the absence of these findings at the end of the treatment period were considered to be evidence of their incidental origin. The mean number of relative eosinophils and the mean methemoglobin level were significantly higher in females previously treated with 1000 mg/kg/day (both p<0.05) when compared with the controls, but these differences remained within ranges of the historical control data and were therefore considered to be incidental.
- Clinical chemistry: In males treated with 1000 mg/kg/day, mean chloride levels were significantly reduced (p<0.01) after 4 weeks when compared with control males, but remained within the range of the historical control values and were considered to be of no toxicological relevance. Creatinine levels were significantly reduced after 4 weeks' treatment in females treated with 200 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.01) when compared with the controls. This finding was not associated with related changes (ie muscle loss), nor did it exceed the ranges of the historical control values and was therefore considered to be incidental. Reduced potassium levels were noted in females treated with 1000 mg/kg/day after 4 weeks' treatment with the test item. This difference attained statistical significance (p<0.01), but remained within the ranges of the historical control values and was therefore considered to be incidental. After 2 weeks' recovery, no differences in the clinical biochemistry values of control or previously test item-treated animals were noted.
- Urinalysis: a dose-related discoloratopn of the urine was noted after 4 weeks'teratemnt in both sexes treated with 200 mg/kg/day and at 1000 mg/kg/day. This finding was considered to indicate the kidney as the excretory pathway of the test item, and not as an adverse finding. The urinary discoloration was reversible after the recovery period. Urinary protein was significantly elevated (p<0.01) in the males and females treated with 1000 mg/kg/day when compared with the controls. Elevated urinary bilirubin in both sexes treated with 200 mg/kg/day (significant in males, p<0.05) and in both sexes treated with 1000 mg/kg/day (significant in both sexes, p<0.01). Despite the dose-response relationship, these changes remained within the ranges of the historical control values and were possibly related to color alterations caused by excreted test item, although this was not confirmed by separate sample spiking. The urinary pH was significantly (p<0.01) more alkaline in females treated with 1000 mg/kg/day, but this change remained within the range of the historical control value and therefore considered to be incidental. After 2 weeks' recovery, a slightly elevated urinary erythrocyte count was noted in the males treated previously with 1000 mg/kg/day, but this was considered to be an incidental changes as no findings were noted after the end of the treatment period.
- Terminal body weights: The terminal body weights of males (recorded immediately prior to necropsy) showed reductions at all dose levels, with statistically significant differences in males treated with 200 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.01). Despite the marked differences, these were not considered to be indicative of a test item-related change, but an incidental selection of the heaviest control males (mean body weight on day 28 for males 1-5: 325 grams) as opposed to selection of smaller males treated with 1000 mg/kg/day (mean body weight on day 28 for males 21-25: 281 grams).
- Organ Weights: After 4 Weeks: In males, the absolute adrenal and absolute spleen weights were significantly reduced (p<0.05) at 1000 mg/kg/day when compared with the controls. These changes were not accompanied by morphological changes and were considered to be incidental. Elevated kidney-to-body weight ratios were seen in males treated with 200 mg/kg/day (p<0.01) and 1000 mg/kg/day (p<0.01), but these differences were not reflected in the kidney-to-brain weight ratios of these males, and therefore considered to be incidental, despite the contrary opinion of the pathologist. The mean absolute epididymide weights were similar in control and test item-treated males after 4 weeks' treatment, but the organ-to-body weight ratios of the males treated with 200 mg/kg/day or 1000 mg/kg/day were significantly elevated (p<0.05 in both groups) when compared with the controls. These differences were considered to be body weight effects and unrelated to the test item. When compared with the control females, no test item-related differences in absolute or relative organ weights were noted. After 6 Weeks: The absolute and relative organ weights of males and females previously treated with the test item were considered to compare favourably with those of the controls.
- Macroscopic Findings: At the end of the treatment and following recovery period, the following test item-related gross lesions were observed in rats at 1000 mg/kg/day: reddish or dark red discoloration of the mucosa of the stomach (two males and two females), of the mucosa of the rectum (nine males and six females), of the kidneys (all animals), of the mucosa of the urinary bladder (eight males and eight females), of the mesenteric lymph node (four males and three females), as well as of the trachea and of the lung (one female). The other macroscopic findings recorded 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. They consisted of renal pelvic dilation (two control females), thickening of the thymus (one male at 50 mg/kg/day), reddish or dark red discoloration of the ovaries (one control female), of the thymus (one male at 1000 mg/kg/day), of the mandibular lymph node (one male treated with 1000 mg/kg/day), as well as dark red foci at the seminal vesicles (one control male).
- Microscopic Findings: There were a few findings which distinguished test item-treated animals from controls: kidneys: Small amounts of pigment in the cytoplasm of renal tubulus cells (without any tissue reaction) were recorded in all test item-treated animal at 1000 mg/kg/day of the main study. The cytoplasmic pigment was of red/brown color and granular to vesicular. Due to its small amount and red color this pigment was better identifiable in slides stained with Papanicolaou (PAP). In all recovery animals (allocation B) this change was also recorded, however in smaller amounts. Lung: Small amounts of pigment-laden macrophages, multifocally in alveoli (without any tissue reaction) were recorded in one single test item-treated animal treated with 1000 mg/kg/day of the main study (female no. 53). The cytoplasmic pigment was of red/brown color and finely granular. Due to its own intrinsic color, this pigment was already identifiable in slides stained with Papanicolaou. In the recovery animals (allocation B), this change was not recorded.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See comment in endpoint summary in IUCLID and "Summary and discussion of repeated dose toxicity" in the CSR.
Critical effects observed:
not specified
Conclusions:
In this OECD 407 repeated toxicity study, the authors conclude that the NOAEL should be 50 mg/kg bw/day based on a slightly lower mean body weights in males treated with 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 resulted in no unscheduled mortality, no clinical signs of toxicological relevance noted during daily or weekly observations or during functional observational battery (including no effects on the grip strength or locomotor activity), no changes in mean daily food consumption, no toxicologically relevant changes in organ weights or ratios, and no toxicologically relevant macroscopical or microscopical findings. Test item-related findings were generally restricted to slightly lower mean body weights in males treated with 200 mg/kg bw/day and 1000 mg/kg bw/day during the treatment period, passive fecal and urine discoloration, as well as minor macroscopical discoloration of some tissues of the excretory pathway. Based on the results of this study, the authors conclude that NOAEL of the test substance is 50 mg/kg bw/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

A 5-day dose range finding oral toxicity study was performed (RCC 2003). Based on the results of this study dose levels of 50, 200 or 1000 mg/kg bw/day are proposed for the subsequent 28-day study with 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 2003). 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 resulted in no unscheduled mortality, no clinical signs of toxicological relevance noted during daily or weekly observations or during functional observational battery (including no effects on the grip strength or locomotor activity), no changes in mean daily food consumption, no toxicologically relevant changes in organ weights or ratios, and no toxicologically relevant macroscopical or microscopical findings. Test item-related findings were generally restricted to slightly lower mean body weights in males treated with 200 mg/kg bw/day and 1000 mg/kg bw/day during the treatment period, passive fecal and urine discoloration, as well as minor macroscopical discoloration of some tissues of the excretory pathway. Based on the results of this study, NOAEL of the test substance was considered to be 50 mg/kg bw/day.

However, the registrant is of the opinion that because the weight loss is only slight and the effect on body weight was reversible during the recovery period these effects are not adverse and the NOAEL should be 200 mg/kg bw. This value is taken forward in the derivation of the DNELs.


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