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Description of key information

Based on the results of this study, a dose level of 100 mg/kg/day is established as NOAEL (no-observed-adverse-effect-level) and 30 mg/kg bw is the NOEL  (no-observed-effect-level) for the test material.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 18, 2008 - Jun 25, 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
according to guideline
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Principles of method if other than guideline:
GLP compliance:
yes (incl. QA statement)
Limit test:
other: Rat, Crl:WI (Han)
Details on test animals or test system and environmental conditions:
Age at delivery 7 weeks
Source: Charles River, Germany

Body weight range at acclimatization
Males: 186 – 213 grams (mean 200 grams)
Females: 150 – 175 grams (mean 161 grams)

For 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

The animals were gang-housed (2 or 3 animals/sex) in type IV Makrolon® cages. They were provided with means to hide. During the short time period between functional observations and motor activity, the animals were kept individually in type III Makrolon® cages.
Softwood granulate served as the cage litter (supplier: ABEDD LAB&VET Service GmbH, 1160 Wien, Austria). The cages were color labeled indicating the different dose groups. The same colors were used for labeling the treatment equipment and the formulation containers.

Food and drinking water offered ad libitum
Diet: Provimi Kliba 3433.0
Drinking water: tap water from mains supply, offered in Makrolon® drinking bottles freshly filled twice a week.


Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 °C; relative humidity range: 30-70 %). There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.
Route of administration:
oral: gavage
corn oil
Details on oral exposure:
The test material was weighed into a tared glass container on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item in the dose formulation. The mixtures were prepared using a magnetic stirrer. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Preparation once per week
Storage @RT

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:


Samples were taken from all dose groups in weeks 1 and 4, once directly after delivery and once at the end of use (under use conditions, altogether 16 samples) for determination of concentrations at the laboratory of Harlan Laboratories Ltd..

Samples were stored at approximately -20°C and sent to the test site on dry ice.

The samples were analysed within Harlan Laboratories, phase C32494, using a gas chromatograph coupled to a flame ionisation detector based on an analytical procedure provided by the sponsor. The test item was used as analytical standard.

Analysed samples were discarded with the study director’s written consent. The following acceptance criteria was applied to analytical results: sample contents should be within a range of ± 15% of nominal.

Duration of treatment / exposure:
Method Oral, by gavage.
Rationale Administration by gavage is a common and accepted route of exposure for studies of this type.
Daily dose levels Group 1: 0 mg/kg body weight
Group 2: 30 mg/kg body weight
Group 3: 100 mg/kg body weight
Group 4: 300 mg/kg body weight

Rationale for Dose Level Selection:
The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats performed for a similar compound, using dose levels
of 0, 100, 300 and 1000 mg/kg/day, resulting in increased liver weights (absolute and relative) at all dose levels.

Dose Volume:
5 mL/kg body weight

Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6 mg/mL/day
Group 3: 20 mg/mL/day
Group 4: 60 mg/mL/day

Duration of Acclimatization Period: 7 days

Duration of Treatment Period: 28 days

Duration of Recovery Period: 14 days
Frequency of treatment:
daily for 28 days
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw
actual ingested
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight: 20 (10 (5m, 5f) test + 10 (5m, 5f) revovery)
Group 2: 30 mg/kg body weight: 10 (5m, 5f)
Group 3: 100 mg/kg body weight: 10 (5m, 5f)
Group 4: 300 mg/kg body weight: 20 (10 (5m, 5f) test + 10 (5m, 5f) revovery)
Control animals:
yes, concurrent vehicle
Details on study design:
According to guideline
Positive control:
Observations and examinations performed and frequency:
Observations/Measurements Time schedule
Appearance and behavior daily
Mortality daily
Motor activity day 28
FOB predose (day 0), day 7, day 28
Body weight once a week
Food consumption once a week
Water consumption twice a week
Hematology week 5 +7
Clinical chemistry week 5 + 7
Urinalysis week 5 + 7

Mortality, the behavior and appearance of each animal were checked twice daily at working days and once at off days, preferably at the same time (s) each day. Symptoms were recorded with the LIMS.

In week 4, one hour after administration, the rats (the numerical first 5 males and 5 females per group) were removed from their home cages and their motor activity was recorded in special motor activity cages over 60 minutes at 5 minutes intervals. The number of movements was evaluated by counting the number of interruptions of photo beams (7 beams on the x-axis, 4 beams on the y-axis). The data was transferred into the LIMS. Assignment of the rats to the individual measurements followed a randomization schedule. On each measuring day, measurements were conducted simultaneously in 10 rats.

Food consumption was determined once a week by weighing the food per cage which had not been consumed.

Each rat was weighed before treatment and thereafter once a week.

A functional observational battery was performed in the numerical first 5 males and 5 females per group before the first exposure, a week thereafter, and in week 4. In week 4, the functional observational battery was performed after the motor activity measurement. Animal numbers and microchips were not identified for the laboratory staff performing the functional observational battery. Therefore, the observers did not know to which treatment
group the rats belong. The following parameters were examined in the cage, during removal of the cage, in the observation arena and during handling: palpebral closure, ease of removal and handling from cages, muscle tone, lacrimation, salivation, piloerection, fur appearance, mobility, arousal, gait, approach response, touch response, click response, tail pinch response, righting reflex, pupil response, raising, raising behavior, defecation and urination (number of fecal boluses, feces consistency, number of urine pools, urine stain size), hind limb foot splay, fore limb and hind limb grip strengths, internal body temperature, catalepsies, posture, vocalization, convulsions, stereotypy, and any abnormalities.

In weeks 5 and 7 approximately 2.5 mL blood were withdrawn retroorbitally under inhalation anesthesia before necropsy from the rats listed below. The blood samples were divided for hematological and clinico-chemical examinations. Before blood sampling the animals were kept in metabolism cages for the collection of urine for approximately 18 hours without food.

Red blood cells (erythrocytes) /pL RBC (a)
Hemoglobin g/dL HGB (a)
Hematocrit % HCT (a)
Mean cell volume fL MCV (a)
Mean hemoglobin content pg MCH (a)
Mean hemoglobin concentration g/dL MCHC (a)
Platelets /nL PLT (a)
Reticulocytes % RET% (a)
Absolute number of reticulocytes /nL RET (a)
White blood cells (leukocytes) /nL WBC (a)
Absolute number of neutrophilic granulocytes /nL NEUT (a, b)
Absolute number of lymphocytes /nL LYM (a, b)
Absolute number of eosinophilic granulocytes /nL EOS (a, b)
Absolute number of basophilic granulocytes /nL BASO (a, b)
Absolute number of monocytes /nL MONO (a, b)
Absolute number of large unstained cells /nL LUC (a, b)
Neutrophilic granulocytes % NEUT% (a, b)
Lymphocytes % LYM% (a, b)
Eosinophilic granulocytes % EOS% (a, b)
Basophilic granulocytes % BASO% (a, b)
Monocytes % MONO% (a, b)
Large unstained cells % LUC% (a, b)
Prothrombin time sec PT sec (c)
Prothrombin time % PT % (c)
Partial thromboplastin time sec PTT (c)

a) ADVIA 120, Siemens Medical Solutions GmbH (Bad Nauheim, Germany)
b) Visual differentiation by a microscope, ZEISS (Oberkochen, Germany)
c) Coasys Plus, Roche Diagnostics (Mannheim, Germany)

Unit Abbreviation Method Instrument
Sodium mmol/L NA ISE, indirect (a)
Potassium mmol/L K ISE, indirect (a)
Calcium mmol/L CA CPC (a)
Chloride mmol/L CL ISE, indirect (a)
Inorganic phosphate mmol/L IP Direct phosphomolybdate (a)
Glucose mmol/L GLUC Glucose-Hexokinase (a)
Urea mmol/L UREA Urease-glutamatic DH (a)
Creatinine μmol/L CREA Jaffé, without deproteinization (a)
Total bilirubin μmol/L TBIL Vanadat-Oxidation (a)
Cholesterol mmol/L CHOL CHOD-PAP (enzymatic) (a)
Triglycerides mmol/L TRIG GPO-PAP (enzymatic) (a)
Bile acids μmol/L BA Enzymatic color (a)
Total protein g/L TP Biuret (a)
Albumin g/L ALB Bromcresol green (a)
Alanine aminotransferase U/L ALAT Kinetic UV, IFCC with P-5-P (a)
Aspartate aminotransferase U/L ASAT Kinetic UV, IFCC with P-5-P (a)
Alkaline phosphatase U/L AP Kinetic color, IFCC AMP (a)
Glutamate dehydrogenase U/L GLDH Kinetic UV, GSCC (DGKC) (a)

a) ADVIA 1650, Autoanalyzer, Siemens Medical Solutions Diagnostics GmbH, (Bad Nauheim, Germany)
b) Clinitek 100, Reflection Spectrophotometer,Siemens Medical Solutions Diagnostics GmbH,
(Bad Nauheim, Germany)
c) Microscope Olympus, BX40F, Olympus Optical CO, LTD, (Hamburg, Germany)
d) Refractometer, Krüss, (Hamburg, Germany)

Sacrifice and pathology:
After 4 Weeks
After 6 Weeks (Recovery)

At the end of the study the rats were anesthetized by a carbon dioxide air mixture and
exsanguinated by opening the abdominal vessels. The rats were necropsied and examined
for gross pathological alterations.

ADRL Adrenal N SC Nerve, sciatic
AORT Aorta OVAR Ovary
BONE Bone OVID Oviduct
BRN Brain PANC Pancreas
DUOD Intestine, small, duodenum PARA Parathyroid
ESOP Esophagus PEYP Peyer's patches
EPID Epididymis PITU Pituitary
EYE Eye PROS Prostate
HRT Heart SALI Salivary gland
I CE Intestine, large, cecum SEMV Seminal vesicle
I CO Intestine, large, colon SKIN Skin
I RE Intestine, large, rectum SPIN Spinal cord
ILEU Intestine, small, ileum SPLN Spleen
JEJU Intestine, small, jejunum STOM Stomach
KIDY Kidney TEST Testis
LIVR Liver THYM Thymus
LUNG Lung THYR Thyroid
M SK Muscle, skeletal TRAC Trachea
MAND Lymph node, mandibular UR Ureter
MAMY Mammary gland URIN Urinary bladder
MARR Bone marrow UTER Uterus
MESE Lymph node, mesenteric VAGI Vagina
N OP Nerve, optic
REPRO, Reproductive organs

Terminal body weight (after exsanguination)
Kidneys (together)
Testes (together)
Ovaries (together)
Adrenals (together after fixation)
Thyroids with parathyroids (together after fixation)
Brain (cerebrum, cerebellum, medulla oblongata after
Epididymides (together)
Seminal vesicles

For the main kill animals organs and tissues were fixed, histotechnically processed and
examined as listed below.

Adrenal (2)
Bone with knee joint (os femoris)
Bone with bone marrow (sternum, femur)
Brain (cerebrum, cerebellum, brain stem)
Eye (2)
Intestine, large Cecum Colon Rectum
Intestine, small Duodenum Jejunum Ileum
Kidney (2)
Liver (left lateral and right medial lobe)
Lung (with mainstem bronchi)
Lymph nodes mandibular (2) mesenteric
Mammary gland (inguinal)
Micro transponder
Muscle, skeletal (thigh)
Nasal turbinates
Nerve, optic (2)
Nerve, sciatic
Parathyroid (2)
Peyer’s Patches
Reproductive organs, female
Ovary (2) Oviduct (2)
Uterus (cornu/corpus/cervix) Vagina
Reproductive organs, male Epididymis (2) Prostate
Seminal vesicle
Testis (2)
Salivary gland (2)
(submandibular, parotid, sublingual)
Skin (inguinal)
Spinal cord (cervical, thoracal, lumbal)
Stomach (proventricular, fundic, pyloric)
Thyroid (2)
Ureter (2)
Urinary bladder
Zymbal's gland (2)
All tissues showing abnormality

Slides of all organs and tissues listed above which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. As histomorphologic changes were observed in the liver of high-dose animals, the livers of the animals of the low- and mid-dose group were also examined.
All parameters were analyzed separately for each sex and time. To take the number of dose
groups into account all the test procedures used maintain a multiple significance level of
a= 0.05.
Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
treatment related foam cell accumulation in the lungs
Histopathological findings: neoplastic:
not examined
Details on results:
During the daily clinical observations, only one female high dose animal showed hair loss within the treatment period. No other symptoms in the animals were noted, the hair loss is not considered to be treatment related.

No test item-related effects have been recorded.

No test item-related differences in absolute or relative food consumption were noted.

The functional observational battery (FOB) was performed in designated animals on day 0, day 7 and day 28 of treatment. No significant changes were noted in the autonomous, neuromuscular, sensomotoric or central nervous domain at any time point. Motor activity was measured in designated animals on day 28. No dose-dependent significant changes of locomotor activity in both genders versus control were noted.

Hematological evaluation at the end of the 28-day treatment period (week 5) and the recovery period (week 7) showed no relevant changes of hematological or coagulation parameters. Slight variations of haemoglobin, haematocrit and the number of erythrocytes were within the normal internal laboratory range of values.

Clinical chemistry at the end of the 28-day treatment period (week 5) and the recovery period (week 7) showed no relevant changes of serum-electrolytes, -substrates, -proteins, and -enzymes. A few parameters showed slight changes that were minimal and within the normal internal laboratory range of values. At the end of the recovery these slight changes could no longer be observed.

Urine weight and specific urinary gravity did not reveal any toxicologically relevant changes. The semi-quantitative urinalysis in week 5 showed that 5 of 10 female group 4 animals showed slight traces of blood which was confirmed by the verification of erythrocytes in the urine sediment. In this treatment group additionally epithelial cells were found in the urine sediment. In week 7 these findings proved to be reversible. No histopathological correlate was found.

Organ weights showed increased absolute liver weights in females of groups 3 (100 mg/kg) and 4 (300 mg/kg) and increased relative liver weights in both sexes of group 4 (300 mg/kg) after 28 days of treatment. The slight absolute liver weight increase in group 3 (100 mg/kg) females is considered a borderline effect. Histopathology of the liver in all treatment groups was inconspicuous.

At gross pathology only spontaneous alterations were observed.

At histopathology, group 4 rats of both sexes (300 mg/kg) exhibited treatment related foam cell accumulation in the lungs. In recovery animals, foam cell-accumulation was not reversible in female rats whereas the lungs of male rats were inconspicuous. Other treatment related organ alterations were not observed.

Key result
Dose descriptor:
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: please refer to the summary
Dose descriptor:
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: please refer to summary
Key result
Critical effects observed:
Based on the results of this study, a dose level of 100 mg/kg/day is established as NOAEL (no-observed-adverse-effect-level) and 30 mg/kg bw is the NOEL (no-observed-effect-level) for the test material.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
100 mg/kg bw/day
Study duration:
Quality of whole database:
Guideline study under GLP conditions
respiratory system: lower respiratory tract

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

Justification for classification or non-classification

Based on the data provided, the test item is not classified for STOT RE according to Regulation (EC) No 1272/2008.