Registration Dossier

Administrative data

Description of key information

In the course of a GLP conform subacute toxicity study, the test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days according to the OECD guideline 407 (RCC Ltd, 2002). Administration of the test item did not induce mortalities, signs of treatment related toxicity or any other changes. A transient, minor decrease in locomotor activity in high dose animals is considered to be a not-treatment related effect. Thus, the NOAEL is considered to be 1000 mg/kg bw/day.

In a 90-day repeated dose toxicity test on an analogue substance (comparable to OECD TG 408, acc. to GLP) by oral gavage in male and female Sprague Dawley rats no toxicity occurred up to the limit dose of 1000 mg/kg bw/day ETH50. The NOAEL was determined to be 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: HanBrhWIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Fuellinsdorf / Switzerland- Age at study initiation: 7 weeks- Weight at acclimatization: Males: 129.6 - 161.0 grams (mean 139.8 grams), Females: 109.7- 130.7 grams (mean 119.1 grams)- Housing: groups of five animals- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 (batch nos. 119/01 and 05/02) rat maintenance diet (Provimi Kliba AG, Kaiseraugst/ Switzerland) was available ad libitum.- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.- Acclimation period: one weekENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 3- Humidity (%): 30-70- Air changes (per hr): 10-15- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
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- Lot/batch no. (if required): 433337/1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item/vehicle mixtures were prepared and about 2 g (weighed to the third decimal place) of each mixture was weighed into a 100-mLvolumetric flask by RCC Ltd, Toxicology Division, Itingen. These samples for analysis of concentration and homogeneity were delivered to the analytical laboratory where they were stored deep-frozen (about -20 °C) until analysis. Storage stability samples were collected after storage (2 hours, 7 days) and then delivered to the analytical laboratory where they were stored deep-frozen until analysis.The delivered samples were dissolved in about 70 mL of acetonitrile/tetrahydrofuran (1-t-l v/v) using an ultrasonic bath. Then, the 100-mL volumetric flasks were filled to the mark with acetonitrile/tetrahydrofuran (1-1-1 v/v). Depending on the dose group, the latter sample solutions were further diluted with acetonitrile/tetrahydrofuran (l-i-l v/v) to yield concentrations within the calibration range. Finally, a defined aliquot was quantified by HPLC.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals; additional five animals per sex were available in groups 1 (controls) and 4 (high dose) for the assessment of reversibility of effects.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon the results of a non-GLP 5-day doserange-finding study (RCC Study Number 842521) in which the test substance was administered by gavage to 2 rats per group and sex.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: dailyDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: weeklyBODY WEIGHT: Yes - Time schedule for examinations: weeklyFOOD CONSUMPTION AND COMPOUND INTAKE:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: YesHAEMATOLOGY: Yes- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period- Anaesthetic used for blood collection: Yes, light isoflurane- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Reticulocyte fluorescence ratios, Nucleated erythrocytes (normoblasts), Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, Thromboplastin time (= prothrombin time), activated partial thromboplastin timeCLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.- Parameters examined: Glucose, Urea, Creatinine, Uric Acid, Bilirubin total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein total, Globulin, Albumin/Globulin ratioURINALYSIS: Yes- Time schedule for collection of urine: at the end of the dosing and the treatment-free recovery period- Metabolism cages used for collection of urine: Yes - Animals fasted: Yes, for approximately 18 hours before sampling- Parameters examined: Volume (18-hour), Specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Nitrite, Urobilinogen, Urine sediment, Red blood cells, White blood cells, Crystals (Triple phosphate)OTHER:GRIP STRENGTH: Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AGF 25N).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: YesHISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as clinical laboratory data:• 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.
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related clinical signs of toxicological relevance were noted during daily or weekly observations (weeks 1-3).
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weight were noted at any dose level tested.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes in food consumption were noted at any dose level tested.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes of toxicological significance were noted after treatment or recovery in the hematology parameters at any dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The clinical biochemistry parameters of the test item-treated rats were similar to those of the controls after the treatment and recovery periods.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis parameters of the test item-treated rats were unaffected after the treatment and recovery periods
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
FUNCTIONAL OBSERVATIONAL BATTERY: No test item-related clinical signs of toxicological relevance were noted during functional observations (week 4).GRIP STRENGTH: No test item-related differences in fore- or hindlimb grip strength values were noted at any dose level tested.LOCOMOTOR ACTIVITY: Slight reductions in mean locomotor activity were noted in both sexes treated with 1000 mg/kg/day. These differences were transient, but contributed to slightly lower total locomotor activity on both sexes and was considered to be a minor effect related to the test item. The locomotor activity values of the rats treated with 50 mg/kg/day or 200 mg/kg/day compared favorably with those of the controls.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related differences in the absolute or relative organ weights were noted at any dose level after four weeks' treatment or two weeks' recovery.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic changes were noted at any dose level after the treatment or recovery periods.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related microscopic changes were noted at any dose level after the treatment or recovery periods.
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
Key result
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:
other: No adverse effects observed at this dose level
Critical effects observed:
no
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-09-28 - 2005-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Note for guidance on repeated dose toxicity. Committee for proprietary medicinal products (CPMP/SWP/1042/99). European Agency for Evaluation of Medicinal Products, 27 July 2000.
Deviations:
no
Principles of method if other than guideline:
Test design comparable with OECD TG 408;
extended to the assessment of the following parameters: T3, T4, TSH, testosterone (males), estradiol and progesterone (females).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Body weight at acclimatization: 214 g (range: 202 g to 232 g) for the males, 155 g (range: 131 g to 171 g) for the females
- Housing: individually housed in suspended wire-mesh cages (43 x 21.5 x 18 cm)
- Diet: A04 C pelleted maintenance diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 8 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hours / 12 hours, light/dark cycle

IN-LIFE DATES:
- From: 2004-10-06 To: 2005-01-07
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % (w/v) carboxymethylcellulose in purified water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder using a mortar and pestle, and then mixed with the required quantity of vehicle in order to achieve the concentrations of 10, 50 and 200 mg/mL, and then homogenized using a magnetic stirrer. The test item dosage forms were prepared on a weekly basis depending on their stability (i.e. 9 days) and were stored at +4° C prior to use and protected from light, according to the storage conditions of the test item.

VEHICLE
- Justification for use and choice of vehicle: solubility of the test item in 0.5 % CMC
- Concentration in vehicle: 10, 50 or 200 mg/mL
- Amount of vehicle: constant dose volume of 5 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations of the test were analysed to check homogeneity, concentration and stability. Actual concentrations of the test item in the dosage forms were determined on weeks 1, 4, 8 and 13.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/d (groups 1,2,3,4, respectively)
Basis:
actual ingested
No. of animals per sex per dose:
Main study: 10 animals (all groups)
Recovery: 6 animals (control and high dose group only)
Toxicokinetics: 6 animals (low, mid and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Results of a 2-week toxicity range finding oral study (CIT/Study No. 28340 TSR):

The administration of the test item by oral route (gavage) to Sprague-Dawley rats for 14 days was well tolerated when given at 300, 600 or 1000 mg/kg bw/day. The only variation was a decrease in the white blood cell and lymphocyte counts in females treated at 1000 mg/kg bw/day. No findings were noted at histopathology.
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Mortality/Viability: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment, and then once a week until the end of the study

FOOD CONSUMPTION:
- Time schedule for examinations: once a week
- Food consumption for each animal determined calculated as g food / animal / d: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time and dose group schedule for examinations: all principal animals before the beginning of the treatment period and on all principal animals on one occasion at the end of the treatment (week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 13: All principal animals from the groups 2 and 3 (low and mid dose group) and the last 10 animals from the groups 1 and 4; at the end of the treatment free period (week 18): The first 6 animals from groups 1 and 4 (control and high dose group)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving main group animals
- Parameters examined: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology: neutrophils, eosinophils, basophils, lymphocytes, monocytes, prothrombin time, activated partial thromboplastin time, fibrinogen
- Other: Blood samples were taken from the orbital sinus of the animals (before the daily treatment)
- As no anaemia was observed, the reticulocyte count was not determined. As no relevant abnormalities were observed during hematological investigations, the bone marrow differential cell count was not determined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- How many animals: determined for all principal animals from the groups 2 and 3 and for the last 10 animals from the groups 1 and 4 during week 13 and for the first 6 animals from groups 1 and 4 at the end of the treatment free period (week 18)
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: see HAEMATOLOGY
- Metabolism cages used for collection of urine: Yes (overnight period of at least 14 hours)
- Animals fasted: Yes
- Parameters examined:
Quantitative: volume, pH, specific gravity; semi-quantitative: proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells; qualitative: appearance, colour

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12
- Dose groups that were examined: all surviving animals
- Functional Observation Battery (FOB), detailed clinical examination; e.g. sensory activity / grip strength / motor activity

OTHER:
HORMONAL ASSAYS:
- Time schedule for collection of blood: week 6, 13 for all principal animals from the groups 2 and 3 and for the last 10 animals from the groups 1 and 4, and at the end of the treatment-free period for estradiol and progesterone determination in females for the first 6 animals from the groups 1 and 4 only
- Animals fasted: No
- How many animals: all surviving main group animals
- Parameters examined: testosterone (in males), estradiol and progesterone (in females), thyroid stimulating hormone (TSH) and thyroid hormones (T3 and T4) (in both sexes).

MONITORING OF ESTRUS CYCLE (determined from a fresh vaginal lavage)
- Time schedule:
- on the day of blood collection of hormonal evaluation (week 6, week 13),
- on each morning for the 2 consecutive weeks preceding the day of blood collection of hormonal evaluation in week 13,
- the 3 days preceding blood sampling for hormone analysis at the end of the treatment-free period.
- How many animals: all surviving main group animals (all principal females)

PLASMA LEVELS OF THE TEST ITEM (SATELLITE ANIMALS)
- Time schedule for collection of blood: weeks 1, 6 and 13 (at the end of the treatment period) as follows:
- day 2 predosing: 24 hours after dosing of day 1,
- weeks 6 and 13: 24 hours after dosing
- How many animals: all surviving satellite group animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, at the end of the treatment or recovery period
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone (NARCOREN) and killed by exsanguination.

The following organ weights were recorded on the scheduled dates of necropsy: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid including parathyroid gland. 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.

All preserved tissue samples, as defined under Histopathology, were processed, embedded and cut at an approximate thickness of approximately 4 micrometers, and stained with hematoxylin and eosin (except testes and epididymides which were stained with hematoxylin/PAS).

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
(i) all preserved tissues of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period: adrenals, aorta, brain (including medulla / pons cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides, esophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum (with Peyers patches), jejunum, kidneys, larynx, liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area, optic nerves, ovaries (with oviducts), pancreas, pituitary gland, prostate, rectum, salivary glands (sublingual and submandibular), sciatic nerves, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, ureters, urinary bladder, uterus (horns and cervix), vagina and macroscopic lesions.
(ii) all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period
Tissues of rats from the treatment-free period were not examined since no target organs were identified.
Statistics:
Body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data:
- Kolmogorov-Lilliefors test, log transformation if necessary; normality of distribution
- Bartlett test (three or more groups) or Fisher test (two groups); homogeneity of variances
- Bartlett test, if not homogenous: Dunn test
- Bartlett test, if homogenous: Dunnett test
- Fisher test, if not homogenous: Mann-Whitney / Wilcoxon test
- Fisher test, if homogenous: Students t-test
Details on results:
CAGE SIDE OBSERVATIONS:
No mortality occurred during the study period.
There were no clinical signs apart from piloerection in 1/10 males receiving 250 mg/kg bw/d and in 3/16 males receiving 1000 mg/kg bw/d, which had always disappeared by week 10. As it was not noted in females and not associated with other findings, this observation was considered to be most probably treatment-related but not to be an adverse effect. There was no treatment-related clinical sign in principal females or in satellite animals.

BODY WEIGHT
The treatment had no effect on body weight or body weight gain, whatever the sex and the treatment period.

FOOD CONSUMPTION
The treatment had no effect on food intake, whatever the sex and the treatment period

OPHTHALMOSCOPIC EXAMINATION
There was no treatment-related finding at ophthalmologic examination.

HAEMATOLOGY / CLINICAL CHEMISTRY
There was no biologically significant treatment-related finding at hematology/biochemistry.
On week 13, low- and high-dose males had moderately lower white blood cell counts when compared to controls (up to -25%), due to significantly lower lymphocyte and monocyte counts. Yet there was a high inter-animal variability (2-fold among controls males) and there was no
difference between the mid-dose and control groups. These changes were therefore considered not to be treatment-related. There was no significant WBC count alteration in females or at the end of the recovery period.

URINALYSIS
There was no biologically significant treatment-related finding at urinalysis.

NEUROBEHAVIOURAL EXAMINATION
Treatment had no effect on neurologic functions in any of the animals in each group and sex.

HORMONAL ASSAYS
The treatment had no influence on T3, T4 and TSH plasmatic levels. There were no biologically significant treatment-related alterations in sexual hormones.

MONITORING OF ESTRUS CYCLE
No disturbance in estrous cycle was recorded during the considered period.

GROSS PATHOLOGY / HISTOPATHOLOGY
Macroscopic examination showed no evidence of treatment related effects.
There was no biologically significant treatment-related change in organ weights and no treatment-related finding in microscopic examination.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The oral administration of the test item to rats up to and including 1000 mg/kg bw/d did not cause any biologically significant test item-related effects.
Critical effects observed:
not specified

CHEMICAL ANALYSIS: The dosage forms stability, homogeneity and achieved concentrations were satisfactory.

TOXICOKINETICS:

When measured on day 2 (24 hours after dosing on Day 1) and on weeks 6 and 13 for groups 2, 3 and 4, which received the test item at 50, 250 and 1000 mg/kg/day, plasma levels of FAT 65'080 were not quantifiable for all animals at any time-point.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across source
Key result
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:
other: no effects up to the limit dose
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

In this GLP conform subacute toxicity study, the test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days according to the OECD guideline 407 (RCC Ltd, 2002). 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, the 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 hematology 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. All animals survived until scheduled necropsy. No test item-related clinical signs, no changes in hematology, urinalysis, food consumption or body weight and no effects on organ weight or any micro/macroscopic changes were noted during daily or weekly observations. Minor reduction in locomotor activity was noted in the first 15 min/1h in both sexes treated with 1000 mg/kg/day. Locomotor activity of the recovery groups was not conducted. Measurements of locomotor activity in rodents serve as preliminary tests to determine motor deficits or anxiety. The test is sensitive to motor dysfunction as well as for neuronal damages. A motor dysfunction or an effect of the substance on the nervous system is expected to cause a constant reduction in locomotor activity and to provoke correlating effects in other parts of the FOB. The reduction in locomotor activity was a transient short term effect, limited to the 1000 mg/kg bw group. A dose response relationship or correlating effects in grip strength or clinical signs were not observed. The change in locomotor activity of the high dose group is, therefore regarded as an unspecific, not-treatment related effect. Based on the results of this study, 1000 mg/kg/day was considered to be the no-observed-adverse-effect-level (NOAEL).

In the sub-chronic repeated dose toxicity study (with an analogue substance) under GLP (CIT 28341 TCR, 2005) three groups of 10 male and 10 female Sprague-Dawley rats were treated by daily oral gavage with non-micronized ETH50 as a suspension in the vehicle (0.5 % [w/v]carboxymethylcellulose in purified water), at dose-levels of 0, 50, 250 or 1000 mg/kg/day for 13 weeks. Six additional males and females were added to control and high-dose groups (0 and 1000 mg/kg bw/day) for a 4 -week post-treatment recovery period. Satellite groups of six males and six females were added to the test item-treated groups for toxicokinetic investigations. Satellite animals were discarded without further examination after the last blood sampling. All study animals were checked for mortality, clinical signs and detailed clinical observations were performed. Functional Observation Battery tests were performed and ody weight/ food consumption was recorded. Ophthalmology, hematological and blood biochemical investigations and urinalysis was performed. Plasma levels of the test item were determined on blood samples taken from satellite animals and T3, T4, TSH and sexual hormones (males: testosterone, females: estradiol and progesterone) levels were measured on blood samples taken. Estrous cycle stages of all principal females were determined from vaginal smears. Designated organs were weighed, macroscopic and histological examinations were performed on selected tissues from controls, high-dose group (0 and 1000 mg/kg bw/day) and on macroscopic lesions.

The oral administration of the test substance to rats up to and including 1000 mg/kg bw/day, followed by a 4-week recovery period, did not cause any biologically significant test substance related effects.

The only clinical sign with dose-related frequency was piloerection in males treated at 250 or 1000 mg/kg/day. This finding was infrequent (1/10 at mid-dose, 3/16 at high-dose) and had always disappeared by week 10. As it was not noted in females and not associated with other findings, this observation was considered to be most probably treatment-related but not to be an adverse effect. There was no treatment-related clinical sign in principal females or in satellite animals.

On week 13, low- and high-dose males had moderately lower white blood cell counts when compared to controls (up to -25%), due to significantly lower lymphocyte and monocyte counts. Yet there was a high inter-animal variability (2-fold among controls males) and there was no difference between the mid-dose and control groups. These changes were therefore considered not to be treatment-related. There was no significant WBC count alteration in females or at the end of the recovery period.

Overall, these effects were not considered to be of toxicological significance. Thus, 1000 mg/kg bw/day is considered to be the no-observed-adverse-effect-level (NOAEL) for ETH50 in rats of both sexes.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) 1272/2008. Based on this data the substance does not need to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.