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Toxicological information

Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-09-28 - 2005-09-07
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable guideline study performed under GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
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.
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:
Limit test:

Test material

Constituent 1
Test material form:
solid: bulk
Details on test material:
Please refer to "Confidential details on test material".

Test animals

Details on test animals or test system and environmental conditions:
- 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.

- 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

- From: 2004-10-06 To: 2005-01-07

Administration / exposure

Route of administration:
oral: gavage
other: 0.5 % (w/v) carboxymethylcellulose in purified water
Details on oral exposure:
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.

- 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:
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
Doses / concentrations
Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/d (groups 1,2,3,4, respectively)
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:
- Time schedule: Once daily
- Mortality/Viability: Once daily

- Time schedule: once a week until the end of the study

- 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

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

- 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.

- 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

- 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

- 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

- 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)

- 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).

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.
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

Results and discussion

Results of examinations

Details on results:
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.

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

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

There was no treatment-related finding at ophthalmologic examination.

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.

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

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

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

No disturbance in estrous cycle was recorded during the considered period.

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.

Effect levels

Dose descriptor:
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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


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.

Applicant's summary and conclusion