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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 26, 2016 - Aug 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
other: Justification for dose selection rationale
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
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
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
Critical effects observed:
not specified
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.
Reason / purpose:
other: Justification for dose selection rationale
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 12 - May 30, 2001
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: MITI 1014
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALSCrj: CD (SD) IGS rats (SPF) were obtained from Charles River Japan, Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan). Animals were quarantined and acclimatized, and healthy animals with favorable weight gains were allocated to groups to ensure homogeneity of mean body weights using body weight-stratified randomization for the study. At the onset of the treatment, the animals were 5 weeks old with body weight ranges of 134.1-153.8 g and 114.7-133.0 g for males and females, respectively. Animals were identified by ear-tagging.HOUSING CONDITIONSThe barrier-system animal room was maintained at a stable temperature (23±2°C) and relative humidity (55±10%) with 10-15 air changes per hour and artificial light-dark cycle of 12-12 hours (light on: 7:00 and light off: 19:00). The rats were housed in hanging stainless steel cages with wire-mesh floor (one rat per cage, 165 Wx300 Dx150 H mm, TOKIWA KAGAKU KIKAI CO., LTD.). Trays were changed twice a week, cages once a week and rack once a month. The racks and cages were identified by individual cards. The animals had free access to an MF pelleted diet (Oriental Yeast, Co., Ltd.) and water (chlorinated) from the Hita City supply via automatic watering system with sipper tubes. The diet and housing materials were autoclaved at 121°C for 30 min prior to use. Analysis of contaminants in both the diet and drinking water confirmed that they would not affect the test system.
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Rationale for dosage selection:A 14-day preliminary repeated-dose oral toxicity study was carried out at three doses of 50, 250 or 1,000 mg/kg/day. One female in the 1,000 mg/kg group died. Abnormalities were noted in blood chemical examinations in the 50, 250 and 1,000 mg/kg groups, in body weights, organ weights and histopathological examinations in the 250 and 1,000 mg/kg groups, and in clinical signs, hematological examination and necropsy in the 1,000 mg/kg group. Therefore, high dose level at 320 mg/kg/day and three lower doses at 80, 20 and 5 mg/kg/day were set for the main study. Recovery groups were prepared for the vehicle control and 320 mg/kg groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC Analytical Conditions(1)Instrument (HP6890)Date analyzer:HP GC-Chemstation (Hewlett packard) Gas chromatograph: HP6890 Series (Hewlett packard)Controller:G1512A (Hewlett packard)Injector:18593B (Hewlett packard)(2)ConditionColumnHP-1 (F.T.0.25 µm) 0.32 mm i.d. x 30 mOven temperature:150°C(0 min)-*20°C /min-X290°C (0 min)Injection temperature:300°CDetector:FIDDetector temperature:300°CInjected amount:1µlInjection method:SplitlessCarrier gas:HeliumCarrie gas flow rate:0.9 mllmin
Duration of treatment / exposure:
Treatment by oral gavege was carried out daily in the morning for 28 days, followed by observation for additional 14 days recovery period. A Nelaton Catheter (TERUMO CORPORATION) and a syringe (TERUMO CORPORATION) were used for dose administration.
Frequency of treatment:
daily for 28 days
Remarks:
Doses / Concentrations:0, 5, 20, 80, 320 mg/kg bwBasis:actual ingested
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight/day: 12 Group 2: 5 mg/kg body weight/day : 6Group 3: 20 mg/kg body weight/day : 6Group 4: 80 mg/kg body weight/day : 6Group 5: 320 mg/kg body weight/day: 12
Control animals:
yes, concurrent vehicle
Positive control:
no
Observations and examinations performed and frequency:
MORTALITY / VIABILITYObservations for mortality/viability were recorded twice daily.GENERAL CAGESIDE OBSERVATIONS (DAILY)The day of the initiation of treatment was defined as day 1, and the preceding day as day -1. The week of the initiation of treatment was defined as week 1. Also, the next day of the final dosing was defined as recovery day 1, and the week of the initiation of recovery as recovery week 1.CLINICAL SIGNSAll animals were observed at least once daily for clinical signs.BODY WEIGHT All animals were weighed as follows:Before Dosing:day -2 (at grouping)During Dosing Period:day 1, 3, 8, 12, 17, 21, 26 and 28During Recovery Period: day 1, 5, 10 and 14In addition, immediately before necropsy, body weights were measured for calculation of relative organ weights.FOOD INTAKEBefore Dosing: Once per dayDuring Dosing period:day 3, 8, 15, 22 and 28During Recovery Period:day 4, 8 and 14HEMATOLOGICAL INFORMATION1) Red Blood Cell Count (RBC)2) White Blood Cell Count (WBC)3) Hemoglobin Conc. (Hb)4) Hematocrit Value (Ht)5) Mean Corpuscular Volume (MCV)6) Mean Corpuscular Hemoglobin (MCH)7) Mean Corpuscular Hemoglobin Conc. (MCHC) 8) Platelet Count (Platelet)9) Reticulocyte Count (Reticulo)10) Prothrombin Time (PT)11) Activated Partial Thrornboplastin Time (APTT) 12) Differentiation of Leukocytes (Stab Neutrophils (N-Band), Segmented Neutrophils (N-Seg), Eosinophils (Eosin), Basophils (Baso), Lymphocytes (Lymph), Monocytes (Mono))CLINICAL BIOCHEMISTRYThe following clinical biochemistry parameters were determined:Parameters1) GOT2) GPT3) Alkaline Phosphatase (ALP)4) LDH5) CPK6) Cholinesterase (ChE)7) y-GTP8) Total Cholesterol (T-Cho)9) Triglyceride (TG)10) Glucose11) Total Protein (T-Protein)12) Albumin13) A/G Ratio14) Blood Urea Nitrogen (BUN)15) Creatinine16) Total Bilirubin (T-Bil)17) Calcium (Ca)18) Inorganic Phosphorus (IP)19) Sodium (Na)20) Potassium (K)21) Chloride (Cl)UrinalysisIndividual urine samples were collected in metabolic cages (200 W X 200 D X 380 Hmm) for 16 hr, at the termination of the dosing (day 28) and recovery (day 14) periods, and examined for volume, color, and additional items of pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen using a test paper (N-Multistix®, Bayer-Medical). Urinary sediment was examined in the vehicle control 80 and 320 mg/kg males and females, and 20 mg/kg females at end of the dosing period, and in the vehicle control and 320 mg/kg males and females at the end of the recovery period.NecropsyAnimals were subjected to a detailed gross necropsy.Organ WeightsFollowing organs were measured in wet weight:Lungs, Liver, Heart, Kidneys, Testes, Ovaries, Brain, Spleen, Adrenals
Sacrifice and pathology:
NECROPSYSacrifice:after 4 weeks 28-July-2006 (groups 1-3, Allocation A)after 8 days 07-July-2006 (group 4, Allocation A and B)after 6 weeks 11-Aug-2006 (group 1, Allocation B)after 6 weeks 01-Sept-2006 (group 3, Allocation B)All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. From group 4 animals (allocation A and B), blood samples were taken by heart puncture at necropsy if possible. No blood for determination of coagulation parameters was collected from these animals.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 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 lesionsAdditional tissues (such as ear tattoo) were retained in accordance with standard operating procedures but were not processed or examined further.ABSOLUTE AND RELATIVE ORGAN WEIGHTSThe following organ weights were recorded on the scheduled dates of necropsy:Brain Thymus Spleen Ovaries Heart Kidneys Testes Liver Adrenals EpididymidesThe 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.HISTOTECHNIQUEAll organ and tissue samples, as defined under Histopathology (following), 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 in boldface type (see Necropsy, above) that were collected at terminal sacrifice from the animals of the control and group 3 were examined by the study pathologist.Since test item-related morphologic changes were detected in organs of the high-dose (group 3) animals, those same organs (liver, adrenal glands, and testes) from the low-dose group (group 2) were examined to establish a no-effect level.The same organs of group 4 animals which were terminated in extremis after 6 days of treatment on day 8 (July, 7th, 2006) were not examined.
Other examinations:
FACTORS THAT MIGHT HAVE AFFECTED THE STUDY1)Unforeseeable circumstances that might have affected the reliability of the study In one female (no. 61) in the 20 mg/kg group, abnormal prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in the hematological examinations at the end of the dosing period. An accelerated coagulation was suggested in this case because of fibrin clot had been formed in the specimen. Given that this change was observed only in one animal in the 20 mg/kg group (the middle dose (1)) with no similar values observed in other animals, this change is likely to be an accidental change rather than a change related to the test article. Accordingly, the PT and APTT values of this animal were excluded from the statistical analysis.The above-mentioned unforeseeable circumstances that might have affected the reliability of the study were considered to have no effects on the results of the study.2)Deviations from the protocolWith respect to the replacement of housing equipment for the housing environment, the protocol stipulated that racks would be replaced at a frequency of every 4 weeks; the rack replacement in week 4 was delayed by 2 days (i.e., once in 30 days). In the present final report, the frequency of the rack replacement is described as once a month.There were no environmental factors that might have affected the reliability of the study results.
Statistics:
Data regarding body weights, food intakes, hematological examinations, blood chemical examinations, urine volume and organ weights were analyzed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the Dunnett's test.If the variances were not homogeneous in the Kruskal-Wallis's test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the nonparametric Dunnett's test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS During Dosing PeriodMale: Salivation was observed in the vehicle control group (7112), 5 mg/kg group (616), 20 mg/kg group (4/6), 80 mg/kg group (416), and 320 mg/kg group (12/12).Clinical aspects of salivationVehicle control group: Sporadically from day 8 to day 28, immediately after dosing.5 mg/kg group:Sporadically or continuously from day 6 to day 28, immediately after dosing.20 mg/kg group:Sporadically from day 6 to day 28, immediately after dosing.80 mg/kg group:Sporadically or continuously from day 6 to day 28, immediately after dosing.320 mg/kg group:Sporadically or continuously from day 6 to day 28, immediately after dosing.Female: Salivation was observed in the vehicle control group (4/12), 5 mg/kg group (616), 20 mg/kg group (4/6), 80 mg/kg group (516), and 320 mg/kg group (10/12). Staining of the hair (1112) were observed in the 320 mg/kg group in week 1.Clinical aspects of salivationVehicle control group: Sporadically from day 6 to day 28, immediately after dosing.5 mg/kg group:Sporadically from day 6 to day 21, immediately after dosing.20 mg/kg group:Sporadically from day 6 to day 28, immediately after dosing.80 mg/kg group:Sporadically from day 6 to day 28, immediately after dosing.320 mg/kg group:Sporadically or continuously from day 6 to day 28, immediately after dosing. During Recovery PeriodNo abnormalities were noted in all groups.BODY WEIGHTS (FIG,1, TABLE 2, ADDENDUM 2) 2.1 During Dosing PeriodNo abnormalities were noted in all groups.During Recovery PeriodNo abnormalities were noted in all groups.FOOD INTAKES During Dosing Period No abnormalities were noted in all groups.During Recovery Period No abnormalities were noted in all groups.HEMATOLOGICAL EXAMINATIONS At Termination of Dosing PeriodMale: Increased WBC was noted in the 80 mg/kg group. Increased WBC and platelet count were noted in the 320 mg/kg group.Female: Increased WBC and a tendency toward increased platelet count were noted in the 320 mg/kg group. Decreased hemoglobin concentration, hematocrit value and APTT were noted in the 320 mg/kg group.At Termination of Recovery PeriodMale: No abnormalities were noted in all groups.Female: Increased reticulocyte count was noted in the 320 mg/kg group. Decreased RBC, hemoglobin concentration, and hematocrit value were noted in the 320 mg/kg group. Increased segmented neutrophils ratio and decreased lymphocytes ratio were noted in the 320 mg/kg group.BLOOD CHEMICAL EXAMINATIONS At Termination of Dosing PeriodMale: Increased creatinine level and tendencies toward increased LDH activity and BUN level were noted in the 320 mg/kg group. Decreased triglyceride level was noted in the 320 mg/kg.Female: Increased GOT and LDH activities, increased BUN, creatinine and inorganic phosphorus levels, and decreased chloride level were noted in the 320 mg/ kg group.At Termination of Recovery PeriodMale: Increased GPT activity and inorganic phosphorus level, and decreased albumin level were noted in the 320 mg/kg group.Female: Increased BUN and creatinine levels, and decreased chloride level were noted in the 320 mg/kg group.URINALYSIS At Termination of Dosing PeriodMale: Increased white blood cells, epithelial cells and casts in the urinary sediment were noted in the 320 mg/kg group.Female: A tendency toward positive reaction for occult blood was noted in the 320 mg/kg group. Increased epithelial cells in the urinary sediment were noted in the 80 mg/kg group. Increased white blood cells, epithelial cells and casts in the urinary sediment were noted in the 320 mg/kg group.At Termination of Recovery PeriodMale: No abnormalities were noted in all groups.Male: Increased white blood cells and casts in the urinary sediment were noted in the 320 mg/kg group.ORGAN WEIGHTS At Termination of Dosing PeriodMale: Increased relative liver weight was noted in the 80 mg/kg group. Increased absolute and relative lung weights and relative liver weight were noted in the 320 mg/kg group.Female: Increased relative liver weight was noted in the 80 mg/kg group. Increased absolute and relative lung, liver and kidney weights and relative heart weight were noted in the 320 mg/kg group. Increased relative brain weight and decreased absolute ovary and adrenal weights were noted in the 320 mg/kg group. At Termination of Recovery PeriodMale: Increased absolute and relative lung weights were noted in the 320 mg/kg group.Female: Increased absolute and relative lung weights and increased relative liver, heart, kidney and spleen weights were noted in the 320 mg/kg group.NECROPSY At Termination of Dosing PeriodMale: Enlargement of the liver (116) was observed in the 80 mg/kg group. Pale change of the lungs (316), enlargement of the liver (516), and enlargement of the mediastinal lymph nodes (316) were observed in the 320 mg/kg group. Blackish region of the mucosa in the glandular stomach was observed in the vehicle control group (216), 5 mg/kg group (116), and 20 mg/kg group (116).Female: Pale change of the lungs (116), enlargement of the liver (616), enlargement (1/6) and pale change (316) of the kidneys, and enlargement of the mediastinal lymph nodes (516) were observed in the 320 mg/kg group.At Termination of Recovery PeriodMale: No abnormalities were noted in all groups.Female: Blackish region of the mucosa in the glandular stomach (116) was observed in the 320 mg/kg group.HISTOPATHOLOGICAL EXAMINATIONS At Termination of Dosing PeriodMale: Hypertrophy of the alveolar lining cells (+, 316 ; ++, 316) in the lungs, centrilobular hypertrophy (+, 116) and increased mitoses (+, 116) of the hepatocytes, focal myocarditis (+, 116), germinal center development (+, 116) and hypertrophy of the paracortex (+, 416) in the mediastinal lymph nodes were observed in the 320 mg/kg group. Necrosis of the fundic mucosa (+, 216) in the glandular stomach, focal myocarditis (±, 116), solitary cyst in medulla (+, 116) in the kidneys were observed in the vehicle control group. Necrosis of the fundic mucosa in the glandular stomach was observed in the 5 mg/kg group (+, 111) and 20 mg/kg group (+, 111). Diffuse lipid droplets (++, 116) and focal necrosis (+, 116) of the hepatocytes were observed in the 80 mg/kg group.Female: Foamy cells, focal proliferation of the atypical pneumocytes, hypertrophy of the alveolar lining cells (+, 516) in the lung, centrilobular hypertrophy (±, 216 ; +, 316) of the hepatocytes, focal myocarditis (+, 316), atypical basophilic tubules (++, 6/6) and mineralization of the cortico-medullary junction (f, 116 ; +316) in the kidney, and hypertrophy of the paracortex (+, 416 ; ++, 116) in the mediastinal lymph node were observed in the 320 mg/kg group. Mineralization in the cortico-medullary junction (±, 116) and solitary cyst in medulla (+, 116) in the kidney were observed in the vehicle control group. Microgranuloma (+, 116) in the liver was observed in the 80 mg/kg group.At Termination of Recovery PeriodMale: Foamy cells (+, 516) and hypertrophy of the alveolar lining cells (+, 216) in the lung, focal myocarditis (+, 116), and hypertrophy of the paracortex (+, 116) in the mediastinal lymph node were observed in the 320 mg/kg group.Female: Foamy cells (+, 616), and hypertrophy of the alveolar lining cells (+, 2/6) in the lung, atypical basophilic tubules (+, 316) and mineralization of the cortico-medullary junction (±, 116; +116) in the kidney, hypertrophy of the paracortex (+, 116) in the mediastinal lymph node, and increased extramedullary hematopoiesis (+, 216) in the spleen were observed in the 320 mg/kg group. Mineralization in the cortico-medullary junction (±, 216 ; +, 116) in the kidney was observed in the vehicle control group. Necrosis of the fluidic mucosa (+, I/1) in the glandular stomach, microgranuloma (+, 116) in the liver and solitary cyst (+, 116) in medulla in the kidney were observed in the 320 mg/kg group.
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, the no-observed effect level (NOEL) of the test material in rats under the present experimental condition was estimated to be 20 mg/kg/day, because increased relative liver weight in males and females, increased WBC and enlargement of the liver in males, and increased epithelial cells in the urinary sediment in females were observed in the animals treated at 80 and 320 mg/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Crl:WI (Han)
Breeder: Charles River, Germany
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: m: 234 (214 – 267) g, f: 166 (152 – 179) g
- Fasting period before study: no
- Housing: IV Makrolon® cages with means to hide
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 40 – 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle
- Concentration in vehicle: 5-100 mg/mL
- Amount of vehicle (if gavage): 1 mL
- Lot/batch no. (if required): -
- Purity: -
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC with FID detection

Mean recoveries
day 0: 84-93%
day 6: 80 - 102 %

Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
LD
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
MD
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
HD
No. of animals per sex per dose:
Control: main: 10 m, 10 f, recovers: 5 m, 5 f
LD: main: 10 m, 10 f
MD: main: 10 m, 10 f
HD: main: 10 m, 10 f, recovers: 5 m, 5 f
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

A structurally similar compound, CCH-23 (CAS: 96624-41-8) was tested in a 28-day repeat-dose oral toxicity study (OECD 407) in HanRcc: WIST (SPF) rats at 30, 100 and 300 mg/kg/d in corn oil. At 300 mg/kg/d a slight decrease in body weight gain, slight to moderate effects on some clinical pathology parameters (e.g. reticulocytes, glucose) was observed. At 100 mg/kg/d some clinical pathology parameters were also affected, but less pronounced. Treatment-related histopathological findings at 300 mg/kg/d consisted of minimal centrilobular hepatocellular hypertrophy in 2 male animals. The histopathological and most clinical pathology findings proved to be reversible. Only some slight effects on clinical pathology parameters were
observed at the end of recovery. Therefore, 300 mg/kg/d was defined as the NOAEL (no adverse effect level) and 30 mg/kg/d as the NOEL (no observed effect level).

Another structurally similar compound, CC-5-V (CAS: 129738-34-7) was tested in a 28-day repeat-dose oral toxicity study (OECD 407) in Crj: CD (SD) IGS (SPF) rats at 5, 20, 80 and 320 mg/kg/d in warm olive oil. At 320 mg/kg/d effects on some clinical pathology parameters (e.g. white blood cells, thromboplastin time, LDH). At 80 mg/kg/d some clinical pathology parameters were also affected, but less pronounced. Treatment-related histopathological findings at 320 mg/kg/d consisted of hyperthrophy of alveolar lining cells in the lung, centrilobular hepatocellular hypertrophy, focal myocarditis and hypertrophy of the paracortex in the mediastinal lymph nodes, increased mitosis of hepatocytes and germinal center development in the mediastinal lymph nodes, foamy cells and focal proliferation of atypical pneumocytes in the lung, atypical basophilic tubules and mineralization of the cortico-medullary junction in the kidney. The histopathological and clinical pathology findings proved to be only partial reversible. Therefore, 20 mg/kg/d was defined as the NOEL (no observed effect level).

Therefore, it was expected for the current study that the high dose level of 100 mg/kg/d over a time period of 90 days would cause mild to moderate systemic toxicity, the low dose level of 5 mg/kg/d was expected to cause no or mild systemic toxicity. The dose of 20 mg/kg/d was used to investigate dose-dependent effects.


- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: yes
- Section schedule rationale (if not random): random

Examinations

Observations and examinations performed and frequency:
Observations/Measurements Time schedule No. of rats/sex/group
Appearance and behavior Daily 10 or 15
Mortality Daily 10 or 15
Detailed Clinical Observations Predose (day -1), weekly 10 or 15
Motor activity Day 77 10
Functional observational battery Day 77 10
Body weight Pre-dose,
there after weekly 10 or 15
Food consumption Weekly 10 or 15
Ophthalmological Investigations Day -1, 84 10 or 15
Hematology (including Coagulation) Week 13 10
Week 17 5
Clinical chemistry Week 13 10
Week 17 5
Urinalysis Week 13 10
Week 17 5
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
To compare the treatment groups with the control group, the following statistical procedures were applied separately for each sex and each measuring point. To take the number of dose groups into account all the test procedures used maintain a multiple significance level of 0.05.

Body weight and body temperature: Dunnett-test (2-sided)
Food consumption: Dunnett-test (2-sided)
Water consumption: Dunnett-test (2-sided)
Clinical pathology parameters (hematology including coagulation, clinical chemistry serum parameters, specific gravity and urine weight): Dunnett-test (2-sided)
Functional observational battery (numerical parameters): Kruskal-Wallis followed by Wilcoxon-test
Motor activity (total counts): Dunnett-test (2-sided)
Dunnett-test (2-sided): Dunnett-test (2-sided)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
for details, see results and tablles below
Mortality:
no mortality observed
Description (incidence):
for details, see results and tablles below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Haematological findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Urinalysis findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
for details, see results and tablles below
Gross pathological findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
for details, see results and tablles below
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Details on results:
for details see "any other information on results"

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver

Any other information on results incl. tables

Clinical Signs

The following symptoms were observed during the study:


0 mg/kg/d
5 mg/kg/d
20 mg/kg/d
100 mg/kg/d
Males
Females
Males Females Males Females Males Females
No of animals
15
15
10
10
10
10
15
15
Symptom

Hair loss
1a
3a 3a -
1a 1a 1a 6a
Animal No
9
43-46
12, 16, 18
-
26
61
88
73-77, 79
Skin, wound scabbed,
- back
- head
1a
(d7 - 27)b
-
-
-
1a
(d39 - 65)b
-
1a
(d80 - 89)b
-
Animal No
9
-
-
-
26
-
88
-
Symptom

Skin, swelling soft
- hindleg, right
-
-
-
-
-
1a
(d20 - 21)b
-
-
Animal No -
-
-
-
-
61
-
-

a: No. of animals with this symptom;b: study days (d) when symptom was observed from earliest to latest observation

Several animals of all dose groups showed hair loss. These findings were without a dose-response relationship and also observed control animals. Therefore, these findings are considered incidental.
Single animals of the 100 and 20 mg/kg/d groups showed skin wounds. These findings were without a dose-response relationship and also observed in a control animal. Therefore, these findings are considered incidental.
One 20 mg/kg/d female showed a soft skin swelling at the right hindleg on days 20 and 21. This finding was observed for a very short time period and without a dose-response relationship. Therefore, this finding is considered incidental.

Detailed Clinical Observations
One 100 mg/kg/d female (animal No. 75) showed vocalization on days -1 (pre-dose), 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70. This observation was already present pre-dose and without a dose-response relationship and therefore considered incidental.
Hair loss was observed in the control group (0 mg/kg/d) in 1 male and 2 females on days 7, 14, 21, 28 and 35, in 2 females on days 42, 49, 56, 63 and 70 and in 3 females on days 77, 84 and 90. At 5 mg/kg/d hair loss was seen in 1 male on days 7, 14, 21 and 35, in 2 males on days 42, 56, 63, 84 and 90 and in 3 males on days 49, 70 and 77. At 20 mg/kg/d hair loss was found in 1 female on days 35, 49, and 56. At 100 mg/kg/d hair loss was observed in 2 females on day 42, in 1 female on days 49, 56 and 63, in 1 male and 1 female on day 70, in 1 male and 4 females on days 77 and 84, in 1 male and 5 females on day 90 and in 1 male on days 98, 105, 112 and 118.
Hair loss is a common background finding in Wistar rats and was observed in control and dose group animals of both sexes without showing a dose-response relationship. Therefore, these findings were considered incidental.
A skin wound was seen in the control group (0 mg/kg/d) in 1 male on day 7. At 20 mg/kg/d skin wounds were observed in 1 male (animal No. 26) on days 42, 49, 56, and 63. At 100 mg/kg/d a skin wound was found in 1 male (animal No. 88) on day 84.
In the 20 and 100 mg/kg/d groups only single animals showed skin wounds. These findings were without a dose-response relationship and also observed in a control animal. Therefore, these findings are considered incidental.

Motor Activity
All motor activity parameters – number of counts, on-time, off-time, total distance, rearing, rearing time – did not show treatment-related differences of dose groups versus control.
 
Additional Motor Activity Parameters
Additional parameters of motor activity – off-time (minutes), on-time (minutes), rearing no., rearing time (minutes) – measured on day 77 did not show treatment-related differences of treatment groups versus control.

Functional Observational Battery (FOB)
In the functional observational battery (FOB) no treatment-related changes in autonomous, sensomotoric, neuromuscular, and central nervous system including body temperature measurements were noted.

Autonomous Nervous System
No significant effects on lacrimation, salivation, pupil response, piloerection, defecation (numberof fecal boluses, feces consistency), urination (number of urine pools, urine stain size), and palpebral closure were seen in any dose group on day 77.

Sensomotoric System
No statistically significant effects on approach response, click response, touch and tail pinch response were seen in any dose group on day 77.

Central Nervous System
No statistically significant effects on ease of removal and handling, arousal, fur appearance, raising behavior, or catalepsies were seen in any dose group on day 77.

Body Temperature
No statistically significant effects on ease of removal and handling, arousal, fur appearance, raising behavior, or catalepsies were seen in any dose group on day 77.

Body Weight (Gain)
Mean body weight (absolute values): day 1 - 118

Dose / [mg/kg/d]
Mean Body Weight / [g]
Day 1
Day 42
Day 90
Day 118

Males
Females
Males Females Males Females Males Females
0
234.4
166.1
366.0
215.2
437.9
236.6
457.6
241.4
5
234.4
166.7
359.6
219.1
425.0
241.4
-
-
20
235.8
166.0
363.2
219.7
429.3
241.8
-
-
100
232.2
165.6
354.4
214.3
422.0
236.2
451.8
242.8


Mean body weight (relative compared to control): Day 1 - 118

Dose / [mg/kg/d]
Mean Body Weight / [%]
Day 1
Day 42
Day 90
Day 118

Males
Females
Males Females Males Females Males Females
0
100.0
100.0 100.0 100.0 100.0 100.0 100.0 100.0
5
100.0 100.4 98.3
101.8 97.1
102.0 -
-
20
100.6 99.9
99.2
101.8
98.0
102.2
-
-
100
99.1
99.7
96.8
99.6
96.4
99.8
98.7
100.6


Mean body weight gain (absolute and relative compared to control): Day 1 - 118

Dose / [mg/kg/d]
Weight Gain / [g]
Weight Gain / [%]
Day 1-90
Day 1-118
Day 1-90
Day 1-118

Males
Females
Males Females Males Females Males Females
0
203.5
70.5
229.9
74.4
100.0
100.0
100.0 100.0
5
190.6
74.7
-
-
93.7
106.0
-
-
20
193.5
75.8
-
-
95.1
107.5
-
-
100
189.8
70.6
221.2
80.4
93.3
100.1
96.2
108.1


Body weight and body weight gain did not show any treatment-related changes at the end of the treatment and recovery period.

Food Consumption

Food consumption: Day 1-28

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 1-7
Day 7-14
Day 14-21
Day 21-28

Males
Females
Males Females Males Females Males Females
0
24.14
16.08
25.10
16.53
23.95
16.90
24.22
16.75
5
24.31
16.77
25.14
16.91
24.41
17.46
23.73
17.10
20
24.19
16.07
24.68
16.57
24.12
17.15
23.62
16.79
100
24.65
16.59
25.12
16.92
24.62
17.24
24.38
17.30


Food consupmption: Day 25-56

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 28-35
Day 35-42
Day 42-49
Day 49-56

Males
Females
Males Females Males Females Males Females
0
24.00
16.50
24.01
15.69
23.75
15.21
23.82
15.26
5
23.55
17.11
23.48
17.13**
22.79
16.43**
23.46
16.37
20
23.35
16.98
23.49
16.47**
22.96
15.99
23.20
16.24*
100
23.55
16.89
23.70
16.81**
23.29
16.34**
23.24
16.63**

**: Dunnett’s test 1% significance level; *: Dunnett’s test 5% significance level

Food consupmption: Day 56-84

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 56-63
Day 63-70
Day 70-77
Day 77-84

Males
Females
Males Females Males Females Males Females
0
24.06
15.25
23.42
14.95
22.71
14.96
22.79
15.08
5
23.41
16.13
22.75
15.88
22.34
15.99*
22.50
15.54
20
23.36
15.61
22.61
15.17
22.51
15.42
22.46
15.37
100
23.46
16.37**
23.16
15.84
22.46
15.45
22.92
15.78

**: Dunnett’s test 1% significance level; *: Dunnett’s test 5% significance level

Food consupmption: Day 84-112

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 84-90
Day 90-98
Day 98-105
Day 105-112

Males
Females
Males Females Males Females Males Females
0
22.80
14.79
23.28
15.18
24.41
15.57
24.09
15.74
5
22.43
15.80*
-
-
-
-
-
-
20
22.42
15.20
-
-
-
-
-
-
100
22.74
15.72*
23.05
15.03
24.54
15.61
24.84
15.38

**: Dunnett’s test 1% significance level; *: Dunnett’s test 5% significance level

Food consumption: Day 112-118

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 112-118

Males
Females
0
24.80
15.73
5
-
-
20
-
-
100
25.14
15.61


Food consumption was slightly increased in females compared to control at 5 mg/kg in week 6-8 (day 35-56), week 11 (day 70-77) and week 13 (day 90-98), at 20 mg/kg in week 6 (day 35-42) and week 8 (day 49-56) and at 100 mg/kg in week 6-9 (day 35-63) and week 13 (day 90-98). These slight increases were without clear dose dependency and no corresponding effects on body weight were observed. Therefore, these changes were considered incidental.

Gross Pathology
Main kill

No treatment-related macroscopic organ lesions were found.

Recovery
No treatment-related macroscopic organ lesions were found.

Body and Organ Weights
Main kill

100 mg/kg
Determination of terminal body weights revealed no treatment-related changes in rats of both genders.
Determination of organ weights revealed in males a tendency to increased absolute liver weights (statistically not significant) and a slight increase of relative liver weights. In females, an increase of absolute and relative liver weights was found.

20 and 5 mg/kg
Determination of terminal body and organ weights revealed no treatment-related changes.The decrease of absolute and relative adrenal weights in females dosed with 20 mg/kg was considered to be arisen by chance.
Summary of discussed absolute and relative organ weights (mean values):


Test groups 1 - 4
Males
Females
Group
1
2
3
4
1
2
3
4
Dose / [mg/kg/d] 0
5
20
100
0
5
20
100
Number of animals
10
10
10
10
10
10
10
10
Body weight / [g]

410
395
398
383
216
217
220
215
Liver
abs. / [g]
10.2
9.9
10.2
10.3
5.6
5.8
5.9
6.7**
rel. / [%] 2.5
2.5
2.6
2.7*
2.6
2.7
2.7
3.1**
Adrenals
abs. / [g] 0.061
0.054
0.057
0.051
0.077
0.071
0.063**
0.069
rel. / [%] 0.015
0.014
0.014
0.013
0.036
0.032
0.029**
0.032

abs. = absolute weight
rel. = relative weight
g = gram
* - Test: Dunnett 5% significance level
** - Test: Dunnett 1% significance level

Number in bold are considered to represent treatment-related changes.


Recovery
Determination of terminal body weights and organ weights revealed no treatment-related changes.

Histopathology
Main kill

At histopathology, a multifocal/focal minimal to mild intracytoplasmic vacuolation of hepatocytes was found in 1/10 (5mg/kg), 3/10 (20mg/kg) and 3/10 (100mg/kg) male rats and 0/10 (5mg/kg), 1/10 (20mg/kg) and 3/10 (100mg/kg) female rats. Incidence was a little bit higher in males than in females. The intracytoplasmic vacuolation of affected hepatocytes was characterized by the presence of multiple mid to large sized optically empty vacuoles. These vacuoles were distributed throughout the cytoplasm around the nucleus and enlarged the affected hepatocytes. Most intracytoplasmic vacuolated hepatocytes were located in periportal and midzonal areas of the liver lobules.

No further treatment-related changes were noted in the organs examined.

Immunohistochemistry results – Anti-Adipophilin

Immunohistochemistry for adipophilin (Anti-Adipophilin, Clone AP125, ProGen Kat.Nr. 610102 (LOT310281); Antibody dilution 0.5μg/ml) showed a prominent positive red immunostaining of the membranes surrounding the mid and large sized cytoplasmic vacuoles in midzonal/periportal
hepatocytes in treated animals.

Positive control: a slide from liver tissue of a Carbon tetrachloride treated rat (no. 69/A2002) and a slide from a rat (no. 182/16-IV013-N0) with known positive adipophilin staining.

Animal number
HE-stained slides
Antibody IHC staining Anti-Adipophilin, Clone AP125
69/A2002 = positive control (centrilobular)
-
+++
Red stained membranes of small and large hepatocellular intracytoplasmicvacuoles (centrilobular)
No. 182/16-IV013-N0 = positive control (periportal)
Vacuolation, hepatocellular, microvesicular, diffuse,marked, centrilobular, pronounced
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
-
Vacuolation, hepatocellular, microvesicular (negative for Anti-Adipophilin)
1 and 4 (control males)
No vacuolation, cytoplasmic hepatocytes present
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
31, 34, 35 (group 4 males)
Vacuolation, cytoplasmic, hepatocytes, focal/multifocal, minimal/mild
+++
Red stained membranes of mid to large sized hepatocellular intracytoplasmic vacuoles (midzonal, periportal)
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
71 (group 4 female)
No vacuolation, cytoplasmic hepatocytes present
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
77, 79 (group 4 females)
Vacuolation, cytoplasmic, hepatocytes, multifocal, minimal and mild
+++
Red stained membranes of mid to large sized hepatocellular intracytoplasmic vacuoles (midzonal, periportal)
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding


Immunohistochemistry for adipophilin showed a prominent positive immunostaining of the membranes surrounding the cytoplasmic vacuoles in hepatocytes. Adipophilin is a component of the surface membrane of lipid droplets (Obert at al., 2007). Enhanced expression of Adipophilin,
as noted here in treated animals, is indicative for increased lipid accumulation in the hepatocellular cytoplasm (also called “fatty change”). Most likely, hepatocytes of treated rats developed mid and large sized intracytoplasmic vacuoles as consequence of metabolic activation/adaption of
hepatocytes (with lipid accumulation) to the test item and the vehicle (100% corn oil).

Incidence summary table of discussed treatment-related findings in the liver:


Test groups 1 - 4, main kill
Males
Females
Group
1
2
3
4
1
2
3
4
Dose / [mg/kg/d] 0
5
20
100
0
5
20
100
Number of animals
10
10
10
10
10
10
10
10
Liver
Vacuolation, cytoplasmic; hepatocytes, multivocal
minimal
1

1



2
mild



2



1
Vacuolation, cytoplasmic; hepatocytes, vocal
minimal








mild


3
1


1




Recovery
100 mg/kg
At histopathology, a focal minimal intracytoplasmic vacuolation of hepatocytes was diagnosed in one male rat of the high dose group.
Incidence summary table of discussed treatment-related findings in the liver:


Test groups 1 - 4, main kill
Males
Females
Group
1
 
 
4
1
 
 
4
Dose / [mg/kg/d] 0


100
0


100
Number of animals
5


5
5


5
Liver
Vacuolation, cytoplasmic; hepatocytes, vocal
minimal







mild



1





All other findings noted were considered to be spontaneous and sporadic in nature.

Discussion Pathology

The test material was administered daily orally by gavage to Crl:WI (Han) rats for a period of 90 days. For the main kill, 10 rats per gender were dosed with 5, 20 or 100 mg/kg test material. 100% corn oil served as control. The treatment period was followed by four weeks of recovery. Reversibility of treatment-related changes was assessed in 5 rats per gender in the control group and 5 rats per gender in the high dose group. All rats survived until their scheduled necropsy date.
At gross pathology, no treatment-related changes were noted in the organs examined of main kill and recovery rats.
Determination of terminal body weights revealed no treatment-related changes in rats of both genders for main kill and recovery rats.
For main kill rats, at a dose level of 100 mg/kg test material, determination of organ weights revealed a tendency to increased absolute liver weights (statistically not significant) and a slight increase of relative liver weights in males and an increase of absolute and relative liver weights in females. No clear-cut histomorphological correlate to these weight changes was found in the liver of main kill rats. Maybe increased liver weights were triggered by metabolic activation/adaption of hepatocytes to the test item and the vehicle (100% corn oil).
At histopathology examination of main kill rats, a multifocal/focal minimal to mild intracytoplasmic vacuolation of hepatocytes was found in some rats of both genders at a dose level of 100, 20 and 5 mg/kg. Males were affected more often than females.
Immunohistochemistry for adipophilin showed a prominent positive immunostaining of the membranes surrounding the cytoplasmic vacuoles in hepatocytes. Adipophilin is a component of the surface membrane of lipid droplets (Obert at al., 2007). Enhanced expression of Adipophilin,
as noted here in treated animals, is indicative for increased lipid droplet accumulation in the hepatocellular cytoplasm of main kill rats, often named “fatty change”. Most likely, hepatocytes of treated rats developed mid size and large intracytoplasmic vacuoles as consequence of metabolic
activation/adaption of hepatocytes (with lipid accumulation) to the test item and the vehicle (100% corn oil).
At histopathology examination of recovery rats, a nearly full recovery of the liver changes was noted in rats of the high dose group. One male still exhibited a focal minimal intracytoplasmic vacuolation of hepatocytes.
The oral administration of 5, 20 or 100 mg/kg of test material for 90 days was considered to be tolerated by the rats, as only minor treatment-related histomorphological liver changes were found in some main kill rats, that were considered non-adverse and rather a sign of metabolic
activation/adaption. Moreover, nearly full recovery was shown after 4 weeks without treatment.


Clinical pathology

Results Hematology and Coagulation

Week 13:

5 mg/kg: BASO (absolute) was increased in males.
20 mg/kg: PT R (%) was decreased and PT R (sec) increased in males. HGB was decreased in females.
100 mg/kg: PT R (%) was decreased in PT R (sec) increased in males. HGB, MCV and PTT were decreased in females.

Week 17:

100 mg/kg: HGB and MCHC were decreased in females.

Results Clinical Chemistry

Week 13:
5 mg/kg: TRIG was decreased in males.
20 mg/kg: CL was increased and TRIG decreased in males.
100 mg/kg: GLUC was increased in both sexes. In males, K and TRIG were decreased and BA and CL increased. In females, ASAT was decreased.

Week 17:
100 mg/kg: In females, GLUC was increased and ALB decreased. UREA was increased in males.

Results Urinalysis
Urinalysis did not show any treatment-related alterations in week 13 and 17.

Disussion Clinical Pathology
Regarding hematology, alterations were slight in degree, not consistent in both sexes and within internal reference ranges. They are not treatment-related. The slight but dose-dependent increase of prothrombin time (PT R (sec)) in 20 and 100 mg/kg males could be indicative for a prolongation of the plasmatic coagulation time. However, the alteration was minimal (< 10%) and not accompanied by further clinico-pathological, clinical or histopathological alterations and therefore considered not treatment-related.
Internal reference intervals given as 2.5 and 97.5 percentiles or *min and max:

Parameter
Unit
15 – 52 weeks


males
females
HGB
g/dL
-
13.6-17.0
MCV
fL
-
50.3-57.5
MCHC
g/dL
-
31.6 - 39.7
BASO
103/µL
0*-0.08*
-
PT% R
%
No data
No data
PT R
sec
No data
No data
PTT
sec
No data
No data



Alterations in clinical chemistry were low in degree, not present in both sexes and within or slightly below or above internal reference intervals. They were not treatment-related. Internal reference intervals given as 2.5 and 97.5 percentiles or *min and max:

Parameter
Unit
15 – 52 weeks


males
females
K
mmol/L
4.50-5.50
-
CL
mmol/L 104.6-108.6
-
GLUC
mmol/L 6.3*-7.9*
3.9-7.2
UREA
mmol/L 4.0-5.6
-
ALB
g/L
-
39.5-46.4
TRIG
mmol/L 0.32-0.98
-
BA
mmol/L 12.2-23.2
-
ASAT
U/L
-
62-99


-No alterations, reference intervals not presented.




Applicant's summary and conclusion

Conclusions:
Daily intravenous treatment with 5, 20 and 100 mg/kg the test material to Wistar (Han) rats was clinically tolerated over 90 days. The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 100 mg/kg/d.
Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 408. Daily intravenous treatment with 5, 20 and 100 mg/kg the test material  to Wistar (Han) rats was clinically tolerated over 90 days. The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 100 mg/kg/d.