Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

The NOAEL was 100 mg/kg bw/day as determined in subacute repeated dose toxicity study according to OECD Guideline 407. The doses 300 and 1000 mg/kg were not tolerated by the male rats and proved to be toxic for the female rats because of premature death especially at 1000 mg/kg, effects on body weight, reduced food consumption, changes in behavioral parameters, influence on a few clinicochemical parameters and histopathological organ lesions..

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
24 August, 2010 - 26 May, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Rat, Crl:WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Age at delivery 8 weeksSource: Charles River, GermanyBody weight range at acclimatization ---------------------------------Males: 249 ( 232 – 263 )gFemales: 180 ( 168 – 200 )gAcclimatization --------------For 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.HOUSING & DIET--------------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 libitumDiet: Provimi Kliba 3433.0Drinking water: tap water from mains supply, offered in Makrolon® drinking bottles freshly filled twice a week.ENVIRONMENTAL CONDITIONSCONDITIONS Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 21 ± 2 °C; relative humidity range: 40-71 %). There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.
Route of administration:
oral: gavage
Vehicle:
other: Miglyol 812 Neutral Oil
Details on oral exposure:
DOSE FORMULATIONThe test item preparation, the test item in Miglyol 812 Neutral Oil, was stable for at least 7 days and homogenous as was shown in a prestudy.Therefore, the preparations were done weekly according to the following method:Appropriate amounts of the test item were weighed in suitable containers and diluted with Miglyol 812 Neutral Oil. All concentrations were dissolved under magnetic stirring in a water bath at 60°C. Thereafter, solutions were filled up with vehicle at 30°C to their final volume. Below 30°C the concentration of 200 mg/mL precipitated. Therefore, the preparations were stirred at 37°C for approximately half an hour (minimum) before administration – during each administration period the preparations were stored at 37 °C. Thereafter, the preparations were stored at room temperature without stirring. Exposure to light was kept to a minimum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONSThis analytical phase was conducted at Harlan Laboratories Ltd., Itingen, Switzerland under GLP-compliant conditions to verify the identity of the test item administered and to determine the content, homogeneity and stability of application formulations.Several application formulations were prepared at the test site and representative analytical samples were collected and dispatched to the test site by courier. The test item concentrations were determined by GC coupled to a FID detector and quantified with the area under the peak.The identity of the test material was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±15% of the nominal content. It was also confirmed that no test item was administered to the control group animals.In conclusion, the results obtained within this phase confirm the correct preparation and storage of application formulations during the conduct of this study.The GC conditions as shown below have been applied:GC: AGILENT 6890Sampling unit: AGILENT 7683Column: ZB 5HT - Phenomenex, 30 m x 0.25 mm,0.25 μm film thicknessCarrier gas: Hydrogen, 2.0 mL/min, constant flowInjection: 1 μL, splitlessDetector: FIDDetector parameters: Gas (mL/min): H2: 40, Air: 400, N2: 30Temperatures: Injector: 275 °CDetector: 325 °COven: 80 °C for 1 minat 20 °C/min to 320 °C320 °C for 8 minRetention time: ca. 9.3 min
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 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 (7d/w)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight: 20 (10 (5m, 5f) test + 10 (5m, 5f) revovery)Group 2: 100 mg/kg body weight: 10 (5m, 5f)Group 3: 300 mg/kg body weight: 10 (5m, 5f) Group 4: 1000 mg/kg body weight: 20 (10 (5m, 5f) test + 10 (5m, 5f) revovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The median lethal dose (LD50) for male and female rats after single oral administration of 2000 mg the test material / kg after an observation period of 15 days was calculated to be > 2000 mg/kg. No clinical effects or organ alterations at gross pathological examination had been observed.A 5-day dose range finding study in rats with oral gavage administration was performed at dose levels of 100, 300, and 1000 mg/kg. Some clinicalsymptoms were observed at 100 mg/kg, however at 300 and 1000 mg/kg no clinical symptoms were observed. Therefore, it was considered that doses up to 1000 mg/kg would be tolerated.For a structural similar test material, the results of a 28 day subacute repeat dose toxicity study in rats treated with 30, 100, and 300 mg/kg were additionally used for the dose selection of the present study. At 300 mg/kg increased numbers of foam cells in the lung, indicative of pneumocytes II / macrophage activation were noted. These foam cells were still visible at the end of the recovery period in female rats. 30 mg/kg and 100 mg/kg of this chemical analogue did not result in any adverse effects. Therefore, 100 mg/kg were considered to be the NOAEL (no adverse observed effect level) and 30 mg/kg the NOEL (no effect level) of this chemical analogue.1000 mg / kg was selected as the high dose because mild to moderate toxic effects were expected. 100 mg/kg and 300 mg/kg were dose-levels to enable a dosecorrelation of effects.- Rationale for animal assignment (if not random): random- Rationale for selecting satellite groups: not assigned- Post-exposure recovery period in satellite groups: control group and high dose group, 2 weeks recovery- Section schedule rationale (if not random): random
Positive control:
no
Observations and examinations performed and frequency:
SCHEDULEObservations/Measurements Time scheduleAppearance and behavior twice dailyMortality twice dailyMotor activity day 28FOB predose (day 0), day 7, day 28Body weight once a weekFood consumption once a weekWater consumption twice a weekHematology week 5 +7Clinical chemistry week 5 + 7Urinalysis week 5 + 7GENERAL CAGESIDE OBSERVATIONSMortality, 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.MOTOR ACTIVITYIn 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 CONSUMPTIONFood consumption was determined once a week by weighing the food per cage which had not been consumed.WATER CONSUMPTIONWater consumption was determined twice a week by measuring the water per cage whichhad not been consumed. The parameter was recorded with the LIM-System.BODY WEIGHTSEach rat was weighed before treatment and thereafter once a week.FUNCTIONAL OBSERVATIONAL BATTERYA 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 treatmentgroup 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.CLINICAL LABORATORY INVESTIGATIONSIn 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.HEMATOLOGYRed 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)Instruments/Methodsa) 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)CLINICAL BIOCHEMISTRYUnit Abbreviation Method InstrumentSodium 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)Instruments/Methodsa) 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:
NECROPSYSacrifice:After 4 WeeksAfter 6 Weeks (Recovery)At the end of the study the rats were anesthetized by a carbon dioxide air mixture andexsanguinated by opening the abdominal vessels. The rats were necropsied and examinedfor gross pathological alterations.ADRL Adrenal N SC Nerve, sciaticAORT Aorta OVAR OvaryBONE Bone OVID OviductBRN Brain PANC PancreasDUOD Intestine, small, duodenum PARA ParathyroidESOP Esophagus PEYP Peyer's patchesEPID Epididymis PITU PituitaryEYE Eye PROS ProstateHRT Heart SALI Salivary glandI CE Intestine, large, cecum SEMV Seminal vesicleI CO Intestine, large, colon SKIN SkinI RE Intestine, large, rectum SPIN Spinal cordILEU Intestine, small, ileum SPLN SpleenJEJU Intestine, small, jejunum STOM StomachKIDY Kidney TEST TestisLIVR Liver THYM ThymusLUNG Lung THYR ThyroidM SK Muscle, skeletal TRAC TracheaMAND Lymph node, mandibular UR UreterMAMY Mammary gland URIN Urinary bladderMARR Bone marrow UTER UterusMESE Lymph node, mesenteric VAGI VaginaN OP Nerve, opticREPRO, Reproductive organsABSOLUTE AND RELATIVE ORGAN WEIGHTSTerminal body weight (after exsanguination)HeartLiverKidneys (together)SpleenThymusTestes (together)ProstateOvaries (together)UterusAdrenals (together after fixation)Thyroids with parathyroids (together after fixation)Brain (cerebrum, cerebellum, medulla oblongata afterfixation)Epididymides (together)Seminal vesiclesHISTOTECHNIQUEFor the main kill animals organs and tissues were fixed, histotechnically processed andexamined as listed below.Adrenal (2)AortaBone with knee joint (os femoris)Bone with bone marrow (sternum, femur)Brain (cerebrum, cerebellum, brain stem)EsophagusEye (2)HeartIntestine, large Cecum Colon RectumIntestine, small Duodenum Jejunum IleumKidney (2)LarynxLiver (left lateral and right medial lobe)Lung (with mainstem bronchi)Lymph nodes mandibular (2) mesentericMammary gland (inguinal)Micro transponderMuscle, skeletal (thigh)Nasal turbinatesNerve, optic (2)Nerve, sciaticPancreasParathyroid (2)Peyer’s PatchesPituitaryReproductive organs, femaleOvary (2) Oviduct (2)Uterus (cornu/corpus/cervix) VaginaReproductive organs, male Epididymis (2) ProstateSeminal vesicleTestis (2)Salivary gland (2)(submandibular, parotid, sublingual)Skin (inguinal)Spinal cord (cervical, thoracal, lumbal)SpleenStomach (proventricular, fundic, pyloric)ThymusThyroid (2)TongueTracheaUreter (2)Urinary bladderZymbal's gland (2)All tissues showing abnormalityHISTOPATHOLOGYSlides 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:
All parameters were analyzed separately for each sex and time. To take the number of dosegroups into account all the test procedures used maintain a multiple significance level ofa= 0.05.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
@1000, and 300 mg/kg bw
Mortality:
mortality observed, treatment-related
Description (incidence):
@1000, and 300 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
@1000, and 300 mg/kg bw
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
@1000 mg/kg bw
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
@1000 mg/kg bw
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
@1000, and 300 mg/kg bw
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
@1000 , and 300 mg/kg bw
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
@1000, 300, and 100 mg/kg bw
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
@1000 , and 300 mg/kg bw
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
@1000 , and 300 mg/kg bw
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
@1000 , and 300 mg/kg bw
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITYNo test item was detected in the formulation samples of the control group. The test material wasdetected in all samples of the test item groups within the predefined acceptance limit of ±15% of the nominal concentrations.Three females of group 4 (1000 mg/kg) were found dead between days 14-19, additional 5females of this group were taken out of the study under moribund condition between day22 and 28. One female of group 3 (300 mg/kg) was found dead on day 18. All malessurvived the treatment and recovery period up to their scheduled sacrifice.Several clinical signs were observed in numerous females in group 4 (1000 mg/kg) andsome females of group 3 (300 mg/kg). They consisted of lowered body temperature (coldto hand), incomplete eyelid closure, piloerection, sunken flanks, reduced spontaneousactivity, and increased respiratory rate. These symptoms are common signs in ratsindicating general discomfort.BODY WEIGHT AND WEIGHT GAINBody weight and body weight gain were significantly decreased in group 4 (1000 mg/kg)and group 3 (300 mg/kg) females compared to control resulting in approximately 25 or 8% less body weight and 41 or 54% less weight gain, respectively. All male treatmentgroups and group 2 females (100 mg/kg) did not show significant effects on body weight.During the recovery period the body weights of the remaining group 4 females (1000mg/kg) recovered partially.FOOD CONSUMPTIONSignificant decreases in food consumption were observed in group 4 (1000 mg/kg) femaleson day 14, 21 and 28. During the recovery the decreases were not observed any more,however, due to mortalities in this group and gender, the number of animals was reducedto 2 surviving females.WATER CONSUMPTIONWater consumption was inconsistently increased or decreased, e.g., water consumptionwas decreased in group 3 females and group 4 animals correlating with their generaldiscomfort.FOBNo treatment-related relevant changes were observed on days 0 and 7 during the functionalobservational battery in the autonomous, sensomotoric, neuromuscular or central nervousdomain. On day 28, a few behavioral changes were noted in group 3 females (300 mg/kg)and group 4 females (1000 mg/kg). However, the changes observed are considered to berelated to the general discomfort of the animals rather than real behavioral effects.Total motor activity over 60 minutes on day 28 showed a significant decrease in group 3(300 mg/kg) females. In group 4 (1000 mg/kg) the decrease was even more pronounced(no significance since animal number was reduced to n=2). Males did not show anysignificant differences between treatment groups.HAEMATOLOGYHematological parameters showed a few mild changes that were all within the internallaboratory ranges and, therefore, not considered to be treatment-related. Coagulationparameters did not reveal any changes compared to control.CLINICAL CHEMISTRY / URINALYSISAt the end of the treatment period, clinical chemistry parameters showed slightly increasedcreatinine and urea values in group 3 (300 mg/kg) females. Urinalysis revealed slightlyincreased evidence of protein- and blood-values (semi-quantitatively) in group 4 (1000mg/kg) males, as well as group 2 (100 mg/kg) and group 3 (300 mg/kg) females.Additionally, slightly increased numbers of epithelium cells in the sediment were observedin group 3 (300 mg/kg) females. The changes were not observed at the end of the recoveryperiod in group 4 (1000 mg/kg) females. As the effects were mild, no histopathologicalcorrelate was seen, and the findings were no longer observed at the end of the recovery,therefore, they are not considered adverse.ORGAN WEIGHTSAbsolute and/or relative liver mean weights were increased in males and/or females of the 100 and 300 mg/kg treatment groups. Inmales, a relative liver weight increase was also noted for the high dose group of 1000mg/kg with no histopathological correlation.GROSS PATHOLOGYDuring necropsy of the female decedents dark or red or pale multifocal discoloration of thelungs was a major observation. Necropsy at the end of the treatment period revealed onlyspontaneous alterations in all dose groups. HISTOPATHOLOGY: NON-NEOPLASTICHistopathology evaluation revealed that 300 and 1000 mg/kg of the test item induced changes in the lungs of all males and females, characterized by minimal to severe granulomatousinflammation of the parenchyma as a major finding. These changes induced mortality orearly sacrifice due to poor general condition in one female given 300 mg/kg and in almostall females given 1000 mg/kg. Moreover, severe centrilobular hepatocellular necrosis wasseen in two females given 1000 mg/kg.The granulomatous inflammation of the lung parenchyma in group 3 (300 mg/kg) andgroup 4 (1000 mg/kg) is considered adverse. The dose of 100 mg/kg induced only minor tomild alveolar epithelium degeneration and macrophages accumulation within the alveoli inall males and females. Due to the low grade of severity and as no indications for agranulomatous inflammatory change were evident compared to the 300 or 1000 mg/kggroups, these lesions are not considered to be adverse.In recovery animals, minimal or slight granulomatous inflammation, alveolar epitheliumdegeneration and macrophages accumulation within the alveoli of the lungs were stillpresent in almost all animals given 1000 mg/kg. There were indications for recoverybecause the inflammatory lung lesions were still present but at a lower grade of severitycompared to the changes observed in main kill rats.OTHER FINDINGS
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:
body weight and weight gain
clinical signs
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
respiratory system: upper respiratory tract
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Conclusions:
A NOEL (no observed effect level) could not be established in the present study. 100 mg/kg are considered to be the NOAEL (no adverse effect level) 300 and 1000 mg/kg were not tolerated by the male rats and proved to be toxic for the female rats because of premature death especially at 1000 mg/kg, effects on body weight, reduced food consumption, changes in behavioral parameters, influence on a few clinicochemical parameters and histopathological organ lesions..
Executive summary:

Purpose

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period. This study should provide a rational basis for toxicological risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Study Design

The test material was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 100, 300 or 1000 mg/kg. A similarly constituted control group received the vehicle, Miglyol 812 Neutral

Oil, and served to generate contemporary control data.

The control and high dose groups consisted of 10 male and 10 female rats each. The low dose (100 mg/kg) and mid dose (300 mg/kg) groups consisted of 5 male and 5 female rats each. At the end of the treatment period, 10 rats (5 males and 5 females) per group were scheduled for necropsy. The remaining rats of groups 1 (control) and 4 (1000 mg/kg) were scheduled for a 2-week recovery period. The rats were gang-housed under conventional conditions at the test facility.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high-dose animals, and all gross lesions from all animals. 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.

Results

No test item was detected in the formulation samples of the control group. The test material was detected in all samples of the test item groups within the predefined acceptance limit of ± 15% of the nominal concentrations.

Three females of group 4 (1000 mg/kg) were found dead between days 14-19, additional 5 females of this group were taken out of the study under moribund condition between day 22 and 28. One female of group 3 (300 mg/kg) was found dead on day 18. All males survived the treatment and recovery period up to their scheduled sacrifice.

Several clinical signs were observed in numerous females in group 4 (1000 mg/kg) and some females of group 3 (300 mg/kg). They consisted of lowered body temperature (cold to hand), incomplete eyelid closure, piloerection, sunken flanks, reduced spontaneous

activity, and increased respiratory rate. These symptoms are common signs in rats indicating general discomfort.

Body weight and body weight gain were significantly decreased in group 4 (1000 mg/kg) and group 3 (300 mg/kg) females compared to control resulting in approximately 25 or 8 % less body weight and 41 or 54% less weight gain, respectively. All male treatment groups and group 2 females (100 mg/kg) did not show significant effects on body weight. During the recovery period the body weights of the remaining group 4 females (1000 mg/kg) recovered partially.

Significant decreases in food consumption were observed in group 4 (1000 mg/kg) females on day 14, 21 and 28. During the recovery the decreases were not observed any more, however, due to mortalities in this group and gender, the number of animals was reduced to 2 surviving females.

No significant changes of body temperature of treated animals versus control were observed at any time point.

Water consumption was inconsistently increased or decreased, e.g., water consumption was decreased in group 3 females and group 4 animals correlating with their general discomfort.

No treatment-related relevant changes were observed on days 0 and 7 during the functional observational battery in the autonomous, sensomotoric, neuromuscular or central nervous domain. On day 28, a few behavioral changes were noted in group 3 females (300 mg/kg) and group 4 females (1000 mg/kg). However, the changes observed are considered to be related to the general discomfort of the animals rather than real behavioral effects.

Total motor activity over 60 minutes on day 28 showed a significant decrease in group 3 (300 mg/kg) females. In group 4 (1000 mg/kg) the decrease was even more pronounced (no significance since animal number was reduced to n=2). Males did not show any significant differences between treatment groups.

Hematological parameters showed a few mild changes that were all within the internallaboratory ranges and, therefore, not considered to be treatment-related. Coagulation parameters did not reveal any changes compared to control.

At the end of the treatment period, clinical chemistry parameters showed slightly increased creatinine and urea values in group 3 (300 mg/kg) females. Urinalysis revealed slightly increased evidence of protein- and blood-values (semi-quantitatively) in group 4 (1000 mg/kg) males, as well as group 2 (100 mg/kg) and group 3 (300 mg/kg) females. Additionally, slightly increased numbers of epithelium cells in the sediment were observed in group 3 (300 mg/kg) females. The changes were not observed at the end of the recovery period in group 4 (1000 mg/kg) females. As the effects were mild, no histopathological correlate was seen, and the findings were no longer observed at the end of the recovery, therefore, they are not considered adverse.

During necropsy of the female decedents dark or red or pale multifocal discoloration of the lungs was a major observation. Necropsy at the end of the treatment period revealed only spontaneous alterations in all dose groups. Absolute and/or relative liver mean weights were increased in males and/or females of the 100 and 300 mg/kg treatment groups. In males, a relative liver weight increase was also noted for the high dose group of 1000 mg/kg with no histopathological correlation.

Histopathology evaluation revealed that 300 and 1000 mg/kg test material induced changes in the lungs of all males and females, characterized by minimal to severe granulomatous inflammation of the parenchyma as a major finding. These changes induced mortality or early sacrifice due to poor general condition in one female given 300 mg/kg and in almost all females given 1000 mg/kg. Moreover, severe centrilobular hepatocellular necrosis was seen in two females given 1000 mg/kg.

The granulomatous inflammation of the lung parenchyma in group 3 (300 mg/kg) and group 4 (1000 mg/kg) is considered adverse. The dose of 100 mg/kg induced only minor to mild alveolar epithelium degeneration and macrophages accumulation within the alveoli in all males and females. Due to the low grade of severity and as no indications for a granulomatous inflammatory change were evident compared to the 300 or 1000 mg/kg groups, these lesions are not considered to be adverse.

In recovery animals, minimal or slight granulomatous inflammation, alveolar epithelium degeneration and macrophages accumulation within the alveoli of the lungs were still present in almost all animals given 1000 mg/kg. There were indications for recovery because the inflammatory lung lesions were still present but at a lower grade of severity compared to the changes observed in main kill rats.

Conclusions

A NOEL (no observed effect level) could not be established in the present study. 100 mg/kg are considered to be the NOAEL (no adverse effect level) 300 and 1000 mg/kg were not tolerated by the male rats and proved to be toxic for the female rats because of premature death especially at 1000 mg/kg, effects on body weight, reduced food consumption, changes in behavioral parameters, influence on a few clinicochemical parameters and histopathological organ lesions..

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study under GLP conditions
System:
respiratory system: upper respiratory tract
Organ:
lungs

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 which are reliable and suitable, the test item is not classified for STOT RE according to Regulation (EC) No 1272/2008.