Registration Dossier

Administrative data

Description of key information

The NOAEL of the test item was determined to be 300 mg/kg bw/day in rats orally administerd for consecutive 28 days (OECD 407 Guideline).

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
Study period:
Jan 05, 2009 - Jul 11, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
T
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Age at delivery 7 weeksBody weight range at acclimatization ---------------------------------Males: 171.4 – 194.4 grams (mean 182.3 grams)Females: 133.5 – 151.5 grams (mean 142.6 grams)Acclimatization --------------Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.HUSBANDRYCONDITIONS 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.ACCOMODATIONIn 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 B0657) rodent maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.WATER Community tap-water from Itingen was available ad libitum in water bottles. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
DOSE FORMULATIONThe 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONSConcentration, 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.Analyses were performed by the Study Scientist (or his/her staff), according to a HPLC analytical method supplied by the Sponsor. Details of the analytical method are documented in the raw data generated by the Study Scientist (and/or his/her staff). Unless otherwise specified, the dose formulations were delivered under ambient conditions. They were either analyzed upon transfer or frozen (-20 ± 5 °C) until analysis. Samples of dose formulations were not discarded without the written authorization of the study director.
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, Harlan Laboratories study C31526, using dose levelsof 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 weightDose Concentrations: Group 1: 0 mg/mL/dayGroup 2: 6 mg/mL/dayGroup 3: 20 mg/mL/dayGroup 4: 60 mg/mL/dayDuration of Acclimatization Period: 7 daysDuration of Treatment Period: 28 daysDuration of Recovery Period: 14 days
Frequency of treatment:
daily for 28 days
Remarks:
Doses / Concentrations:0, 30, 100, 300 mg/kg bwBasis:actual ingested
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight: 10 (5 test + 5 revovery)Group 2: 30 mg/kg body weight: 5Group 3: 100 mg/kg body weight: 5Group 4: 300 mg/kg body weight: 10 (5 test + 5 revovery)
Control animals:
yes, concurrent vehicle
Details on study design:
According to guideline
Positive control:
no
Observations and examinations performed and frequency:
MORTALITY / VIABILITYObservations for mortality/viability were recorded twice daily.GENERAL CAGESIDE OBSERVATIONS (DAILY)The animals were observed for clinical signs once daily during the acclimatization period, twicedaily on days 1-3; as well as once daily on days 4-28 of the treatment period and once dailyduring days 29-42 (recovery).DETAILED CLINICAL OBSERVATIONS (WEEKLY)The animals were observed in their home cages, outside their home cages in a standard arenaand in the hand. These observations were performed in random sequence once beforecommencement of administration and once weekly (weeks 1-3) thereafter.FOOD CONSUMPTIONThe food consumption was recorded once during the pretest period and weekly thereafter,using an on-line electronic recording system consisting of a Mettler balance connected to theRCC computer.BODY WEIGHTSBody weights were recorded weekly during pretest, treatment and recovery and beforenecropsy, using an on-line electronic recording system consisting of a Mettler balanceconnected to the RCC computer.FUNCTIONAL OBSERVATIONAL BATTERYDuring week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.GRIP STRENGTHForelimb and hind limb grip strength measurements were performed using a push-pull straingauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangulargrasping ring and with the hind paws outside a triangular grasping ring. Using one hand, theanimals were held towards the base of the tail and steadily pulled away or towards the ring untilthe grip was broken. Each measurement was repeated three times, the means were calculatedand recorded.LOCOMOTOR ACTIVITYLocomotor (decreased or increased) activity was measured quantitatively with AMS FöhrMedical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals weremonitored during the fourth treatment week for a 60-minute period and the total activity of thistime period was recorded.Low beams count was reported in 10-minute intervals as well as the total activity of themeasuring period.CLINICAL LABORATORY INVESTIGATIONSBlood and Urine Sampling: After 4 Weeks: 16-Feb-2009 (Satellite A and B)After 6 Weeks: 02-Mar-2009 (Satellite B) Blood samples were drawn from the retro-orbital plexus from all animals under light isofluraneanesthesia. The animals were fasted in metabolism cages for approximately 18 hours beforeblood sampling but allowed access to water ad libitum. The samples were collected early in theworking day to reduce biological variation caused by circadian rhythms. Blood samples weredrawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolismcage.The assays were performed at Harlan Laboratories Ltd. (R. Draheim, Füllinsdorf / Switzerland)under internal laboratory quality control conditions to assure reliable test results.HEMATOLOGYThe following hematology parameters were determined:Erythrocyte countHemoglobinHematocritMean corpuscular volumeRed cell volume distribution widthMean corpuscular hemoglobinMean corpuscular hemoglobin concentrationHemoglobin concentration distribution widthReticulocyte countReticulocyte maturity index (low, medium,high fluorescence)Leukocyte count, totalDifferential leukocyte count:NeutrophilsEosinophilsBasophilsLymphocytesMonocytesLarge unstained cellsPlatelet countMethemoglobinHeinz bodies (slides were prepared but not evaluated since no changes were seen in the methemoglobin levels)Prothrombin time (= Thromboplastin time)Activated partial Thromboplastin timeCLINICAL BIOCHEMISTRYThe following clinical biochemistry parameters were determined:GlucoseUreaCreatinineBilirubin, totalCholesterol, totalTriglyceridesPhospholipidsAspartate aminotransferaseAlanine aminotransferaseLactate dehydrogenaseGlutamate dehydrogenaseCreatine kinaseAlkaline phosphataseGamma-glutamyl-transferaseSodiumPotassiumChlorideCalciumPhosphorus inorganicProtein, totalAlbuminGlobulinAlbumin/Globulin ratioBile acidsURINALYSISThe following urinalysis parameters were determined:Volume (18 hours)Specific gravity (relative density)ColorAppearancepHNitriteProteinGlucoseKetonesUrobilinogenBilirubinErythrocytesLeukocytes
Sacrifice and pathology:
NECROPSYSacrifice:After 4 Weeks: 16-Feb-2009 (Satellite A)After 6 Weeks (Recovery): 02-Mar-2009 (Satellite B)All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopicalabnormalities were recorded. All animals were anesthetized by intraperitoneal injection ofpentobarbitone and killed by exsanguination.Samples of the following tissues and organs were collected from all animals at necropsy and,unless otherwise indicated, fixed in neutral phosphate buffered 4% formaldehyde solution.Additional tissues (such as ear tattoo) were retained in accordance with standard operatingprocedures but will not be processed or examined further.Adrenal glandsAortaBone (sternum, femur including joint)Bone marrow (femur)Brain (at least 3 levels)CecumColonDuodenumEpididymides (fixed in Bouin's solution)EsophagusEyes w/optic nerve (fixed in Davidson's solution)Harderian gland (fixed in Davidson's solution)HeartIleum, with Peyer's patchesJejunum with Peyer's patchesKidneysLarynxLacrimal gland, exorbitalLiverLungs, filled w/formalin at necropsyLymph nodes - mesenteric, mandibularMammary gland areaNasal cavityOvariesPancreasPituitary glandProstate gland (incl. coagulating gland)RectumSalivary glands - mandibular, sublingualSciatic nerveSeminal vesiclesSkeletal muscleSkinSpinal cord - cervical, midthoracic, lumbarSpleenStomachTestes (fixed in Bouin's solution)ThymusThyroid (incl. parathyroid gland, if possible)TongueTracheaUrinary bladder, filled w/formalin at necropsyUterusVaginaGross lesionsABSOLUTE AND RELATIVE ORGAN WEIGHTSThe organs from allocation A and B animals were weighed before fixation and recorded on the scheduled dates of necropsy. Paired organs were weighed separately. The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.HISTOTECHNIQUEAll organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.HISTOPATHOLOGYSlides 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.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity,body weight, clinical laboratory data, organ weights, and ratios:• The Dunnett-test (many to one t-test) based on a pooled variance estimate wereapplied if the variables could be assumed to follow a normal distribution for thecomparison 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 whenthe data can not be assumed to follow a normal distribution.• Fisher's exact-test was applied to the macroscopic findings.• Student’s t-test was applied to grip strength and locomotor activity data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight effect of treatment with the test item on the body weight gain could not be excluded in animals treated with 300 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
slight effects depending on conditions
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
slight to moderate effects depending on conditions
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):
liver and kidney
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
liver and kidney
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
reversible liver hyperthrophy
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITYNo clinical signs distinguished test item-treated from control animals.BODY WEIGHT AND WEIGHT GAINA slight effect of treatment with the test item on the body weight gain could not be excluded inanimals treated with 300 mg/kg/day.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)No test item-related differences in absolute or relative food consumption were noted.HAEMATOLOGYAfter 4 weeks of treatment, the following statistically significant differences (compared to controls) in hematology parameters were considered to be test item-related:• Slightly decreased hemoglobin and hematocrit in males and females treated with 300 mg/kg/day• Slightly increased hemoglobin concentration distribution width in males treated with 300 mg/kg/day• Slightly decreased relative number of low fluorescence reticulocytes and increased relative number of high fluorescence reticulocytes in females treated with 300 mg/kg/day At the end of the recovery period, no significant changes were noted in males. In females treated with 300 mg/kg/day, slightly decreased mean cell hemoglobin concentration (-1.7%, p<0.01) and decreased hemoglobin concentration distribution width (-9.0%, p<0.05) were noted. The same changes had not been present in the same sex after 4 weeks of treatment. Thus, changes noted in hematology parameters were considered not to be adverse.CLINICAL CHEMISTRYAfter 4 weeks of treatment, the following statistically significant differences (compared to controls) in clinical chemistry parameters were considered to be test item-related:• Moderately decreased glucose level in males treated with 300 mg/kg/day• Slightly increased urea concentration in males treated with 300 mg/kg/day• Slightly increased creatinine concentration in males and females treated with 300 mg/kg/day• Slightly increased cholesterol levels in females treated with 100 or 300 mg/kg/day• Moderately increased triglyceride levels in females treated with 300 mg/kg/day• Moderately increased phospholipid levels in females treated with 100 or 300 mg/kg/day• Slightly increased sodium concentration in males treated with 300 mg/kg/day• Slightly decreased phosphorus concentration in males treated with 300 mg/kg/dayA slightly decreased total bilirubin level (p<0.05) was present in males treated with100 mg/kg/day only and was thus considered not to be test item-related.After the recovery period, a slight increase in glucose levels (+42.7%, p<0.01) as well as a slightdecrease in phosphorus concentration (-13.2%, p<0.01) attained statistical significance in malestreated with 300 mg/kg/day.URINALYSISNo test item-related differences in urinalysis parameters were noted.NEUROBEHAVIOURDuring detailed observation in week 4, increased activity was noted in one control male. No further abnormalities were noted.Grip StrengthNo significant differences were noted in the mean grip strength of test item-treated animals compared to control animals.Locomotor ActivityNo test item-related findings in locomotor activity were noted.ORGAN WEIGHTSAfter 4 weeks of treatment, increased absolute and relative liver weights in females treated with 100 or 300 mg/kg/day and liver to body weight ratio in males treated with 100 or 300 mg/kg/day and increased absolute kidney weight and kidney to body weight ratio in females treated with 300 mg/kg/day the were considered to be test item-related. No differences in organ weights were noted after the recovery period.GROSS PATHOLOGYNo test item-related macroscopic findings were noted.HISTOPATHOLOGY: NON-NEOPLASTICMicroscopically, minimal hepatocellular hypertrophy, mainly centrilobular, was recorded in two males treated with 300 mg/kg/day. This finding correlated with a statistically significant increase in liver to body weight ratio. It showed complete regression after the treatment-free recovery period. The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.HISTOPATHOLOGY: NEOPLASTIC (if applicable)HISTORICAL CONTROL DATA (if applicable)OTHER FINDINGS
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
Based on the results of this study, a dose level of 300 mg/kg/day is established as NOAEL (no-observed-adverseeffect-level) for the test material.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study under GLP conditions

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 the Regulation (EC) No 1272/2008.