Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-15 to 2007-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
27 July 1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
30 September 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): T002675, TIC876
- Physical state: liquid
- Appearance: colorless to yellowish liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 603T-1
- Expiration date of the lot/batch: 2007-12-19
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (15-25 °C) in the original container away from direct sunlight
- Solubility and stability of the test substance in the solvent/vehicle: Stable under storage conditions

OTHER SPECIFICS:
- purity: 97.76%

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanRcc:WIST (SPF)
Details on species / strain selection:
Rationale: recognized by the international guidelines as the recommended test system
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Laboratory Animal Services, CH-4414 Füllinsdorf, Switzerland
- Age at delivery: 7 weeks
- Weight at acclimatization: 160-173 g (males), 134-145 g (females)
- Fasting period before study: no data
- Housing: standard housing conditions; in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet (e.g. ad libitum): ad libitum, pelleted standard Provimi Kliba 3433 (Batch no. 89/06) rat maintenance diet
- Water (e.g. ad libitum): ad libitum, community tap-water from Itingen was available in water bottles
- Acclimation period: Seven days under test conditions following a health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hrs fluorescent light / 12 hrs dark cycle


IN-LIFE DATES: From: 2007-03-15 To: 2007-05-03

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Method: oral gavage
Frequency: daily
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (15-25°C). Homogeneity of the test substance in the vehicle was maintained during daily administration using a magnetic stirrer.
- Dose formulations were prepared weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 130022563006152 and 127487712006075
- Purity: no data
- Storage conditions: at room temperature (15-25°C) in the original container in the dark
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration, 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. The analyses were performed by RCC Ltd according to a GC HPLC TLC method.

Method:
- Application formulations were prepared for each dose group at the test facility. Representative samples were derived by weighing amounts of 2 g accurately into volumetric glass flasks. Samples for content and homogeneity determination were collected on each occasion from the top, middle and bottom of the mixing beakers. Stability samples were taken from each beaker on the first occasion and kept under storage conditions for 2 hours and for 7 days. At the appropriate time-points, samples were dispatched to the test site internally and stored frozen at approximately –20 °C until analysis.
- The samples received were dissolved in methanol by sonication for 5 minutes and then diluted to volume with methanol. Where necessary, sample solutions were diluted with methanol into the calibration range.

- Method of separation: Gas Chromatography (GC)
- Apparatus: Agilent Chemstation Version 6890
- Column: DB WAX, 30 m x 0.53 mm, 1 µm film thickness
- Carrier gas: Helium, 5 mL/min, constant pressure
- Temperatures: Injector: 230 °C
- Detector: 250 °C
- Oven: 100 °C for 3 min at 20 °C/min to 180 °C; 180 °C for 0 min; at 5 °C/min to 220 °C; 220 °C for 5 min
- Injection: 1 µL, splitless
- Detector: FID

- Injected samples were quantified by comparing peak areas of the test substance with reference to the calibration curve. The latter was obtained by correlation of the peak areas of the working standards with their corresponding concentrations (µg/mL), using a linear regression model.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control animals treated with the vehicle PEG 300 only)
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Group 5 (added to the study as the low dose group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
Group 4 (removed from the study). Any raw data collected was not reported but retained in the raw data.
No. of animals per sex per dose:
5 males and 5 females per group (groups 1-5); 50 animals in total
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon the results of a non-GLP, 5-day dose-range finding study (RCC Study Number B21587) in which the test substance was administered by gavage to 2 rats per group and sex.
- Rationale for treatment: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Rationale for animal assignment (if not random): A computer-generated computer algorithm was used to randomly assign animals to test or control groups.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- mortality/viability: twice daily
- general clinical observations: once before commencement of administration, twice daily on days 1-3, as well as once daily on days 4-28

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in random sequence, once before commencement of administration and once weekly (weeks 1-3) thereafter
- Examinations: animals were observed in their home cages, outside their home cages in a standard arena and in the hand

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during acclimatization, treatment and before necropsy. Additionally body weights of moribund animals of group 3 (150 mg/kg bw) were recorded daily from day 8 of treatment period onwards until premature necropsy.
- Examination: using an online electronic recording system consisting of a Mettler balance connected to the RCC computer

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded once during the acclimatization period and weekly thereafter, using an online electronic recording system consisting of a Mettler balance connected to the RCC computer.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks, early in the working day to reduce biological variation caused by circadian rhythms, drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Anaesthetic used for blood collection: Yes, light isoflurane anesthesia
- Animals fasted: Yes, for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all animals
- Parameters examined included: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, platelet (thrombocyte) count, reticulocyte count, reticulocyte maturity index, methemoglobin, total leukocyte count, differential leukocyte count, coagulation: thromboplastin time and activated partial thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks, early in the working day to reduce biological variation caused by circadian rhythms, drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Anaesthetic used for blood collection: Yes, light isoflurane anesthesia
- Animals fasted: Yes, for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all animals
- Parameters examined included: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatinine kinase, alkaline phosphatase, gamma-glutamyl transferase, sodium, potassium, chloride, calcium, phosphorous inorganic, albumin, total protein, globulin and the albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all groups
- Battery of functions tested:
- a modified Irwin screen test : appearance (piloerection, salivation, hunched posture); motor (ataxia, tremor/twitching, prostration, circling, spasm); behavior (hyperactivity, somnolence, increased exploration, reduced grooming, vocalization); respiration (dyspnea, tachypnea, bradypnea); reflexes (blink, pinna, iridic light reflex, push-off (hind leg), pain response, startle/hearing, righting reflex); miscellaneous (lacrimation, limbs cyanotic, mydriasis, miosis, exophthalmos, reduced muscle tone);
- grip strength: forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge. The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.
- locomotor activity: locomotor (decreased or increased) activity was measured quantitatively at the 4th treatment week. Animals residing in the study at this timepoint were monitored for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.


OTHER: The assays of blood parameters were performed at RCC Ltd. (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.
Sacrifice and pathology:
NECROPSY
- Sacrifice after 4 weeks (groups 1, 2, 5).
- All moribund animals in the high dose group (group 3) treated with 150 mg/kg body weight/day were successively killed in extremis prior to the scheduled necropsy date.
- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.
- All animals surviving to scheduled necropsy and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: Yes
- Descriptions of all macroscopic abnormalities were recorded. Samples of tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution.
- organs examined: adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain (4 levels), cecum, colon, duodenum, epididymides (fixed in Bouin's solution), esophagus, eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), heart, ileum with Peyer's patches, jejunum with Peyer's patches, kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), mammary gland area, nasal cavity, ovaries, pancreas, pituitary gland, prostate gland (including coagulating gland), rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes (fixed in bouin's solution), thymus, thyroid (incl parathyroid gland), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina, gross lesions
- Organ weight:
- Recorded in animals surviving to the scheduled dates of necropsy: brain, heart, liver, thymus, kidneys, adrenals, spleens, testes, epididymides, and ovaries.
- The organ-to-terminal body weight ratio, as well as organ-to-brain weight ratios were determined.

HISTOPATHOLOGY: Yes
- Organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 to 4 µm, and stained with hematoxylin and eosin
- Slides of the following organs and tissues were collected at terminal sacrifice from the animals and examined by light microscopy: adrenal glands, bone marrow (femur), brain (4 levels), cecum, colon, duodenum, epididymides (fixed in Bouin’s solution), heart, ileum with Peyers patches, jejunum with Peyer’s patches, kidneys, liver, lungs (infused with formalin), lymph nodes (mesenteric, mandibular), ovaries, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid (incl. parathyroid gland), trachea, urinary bladder (infused with formalin), uterus, vagina and gross lesions.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory investigations, organ weights and ratios and macroscopic findings:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison 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 when the data cannot be assumed to follow a normal distribution.
- Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related findings were noted during daily and weekly (weeks 1-3) observations in all animals treated with 150 mg/kg bw/day (visible weight loss, hunched posture, ruffled fur, decreased activity, abnormal gait, half-closed eyes and breathing noises) and during daily observations in one female treated with 50 mg/kg bw/day (visible weight loss).
Mortality:
mortality observed, treatment-related
Description (incidence):
- All animals except the animals treated with 150 mg/kg body weight/day survived until scheduled necropsy.
- All animals treated with 150 mg/kg body weight/day were killed in extremis successively between day 9 and 18 of treatment period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related changes were noted in males and females treated with 50 and 150 mg/kg bw/day.
- In animals of both sexes treated with 50 mg/kg bw/day, a slight reduction in the mean absolute and relative body weights was noted from day 15 of treatment onwards.
- In animals of both sexes treated with 150 mg/kg bw/day, a severe reduction in the mean absolute and relative body weights was noted from day 8 of treatment onwards and resulted in a successive moribund status of the animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related reduction in the mean and absolute food consumption of males and females treated with 50 and 150 mg/kg body weight/day was noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- No findings were noted in the mean hematology parameters of males and females treated with 50 mg/kg bw/day. Test substance-related changes were noted in animals treated with 15 mg/kg bw/day.
- In animals treated with 15 mg/kg bw/day, elevated red blood cell count, elevated haematocrit, decreased mean corpuscular hemoglobin and hemoglobin concentration (and a few other findings) were noted compared to controls. The findings in red blood cell parameters correlated with marginal elevated hemopoiesis in liver and/or spleen and elevated total bilirubin and were considered to be test item-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Test substance related adverse changes in clinical biochemistry parameters were noted in animals treated with 15 and 50 mg/kg bw/day.
- In animals treated with 15 and 50 mg/kg bw/day, elevated total bilirubin was noted in both sexes compared to controls with statistical significance of p<0.05 in females treated with 15 and 50 mg/kg bw/day, and in males treated with 15 mg/kg bw/day.
- Total bilirubin values of test item-treated animals predominantly stayed slightly above the limits of historical reference data.
- In animals treated with 15 mg/kg bw/day, the finding of elevated bilirubin correlated with slightly elevated hemopoiesis in liver and/or spleen, changes in red blood cell count, and increased liver weights in males.
- In animals treated with 50 mg/kg bw/day, the finding of elevated bilirubin correlated with the microscopic finding of fatty change in the liver.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- No test substance-related findings were noted during the functional observational battery (week 4) in animals treated with 15 or 50 mg/kg bw/day.
- Grip Strength: No test substance-related chagnes in the mean grip strengh were noted at animals in the 15 mg/kg bw/day group. Females treated with 50 mg/kg bw/day showed significantly reduced mean forelimb grip strength values (p<0.05) compared to controls. These differences were considered to be test substance-related.
- Locomotor activity: No test substance-related changes in the mean locomotor activity were noted at animals in the 15 mg/kg bw/day group. Animals treated with 50 mg/kg bw/day showed significantly reduced mean locomotor activity values at some timepoints and in the mean total (all p<0.05). These changes were considered to be test substance-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- In males treated with 15 and 50 mg/kg bw/day, absolute and relative liver weights were elevated. This finding correlated with elevated bilirubin values in both sexes at both dose groups, and the microscopic finding of fatty change in the liver of animals treated with 50 mg/kg bw/day. These changes were considered to be test substance-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Treatment related macroscopic findings were liquid contents of the gastrointestinal tract (5 males, 3 females) and a reduced size of spleen (one female) in the 150 mg/kg bw/day group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Toxicologically relevant microscopic findings were increased fatty change in animals treated with 50 mg/kg bw/day; increased fatty change, apoptosis, hypertrophy and pigment deposits in the liver of animals treated with 150 mg/kg bw/day; and atrophy of spleen and thymus at 150 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
No further data

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
other: Test item-related changes in animals treated with 15 mg/kg body weight/day, such as elevated bilirubin, changes in the red blood cell count and elevated absolute and relative liver weights in males were considered to be adverse.
Remarks on result:
other: no NOAEL could be derived

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

The administered doses of the test substance were confirmed by analysis of the dose formulations, using an analytical method provided by the sponsor. The test substance content in all samples was found to be within the accepted range of ±20% of the nominal concentration. In addition, the homogeneous distribution of the test substance in PEG 300 was demonstrated. The application formulations were considered to be stable for at least 2 hours and 7 days when kept under storage conditions.

Applicant's summary and conclusion

Conclusions:
The oral administration of T002675 to Wistar rats at doses of 15, 50 and 150 mg/kg bw/day for 28 days resulted in adverse changes of different test parameters in all dose groups. Test item-related changes were also observed at the lowest test dose of 15 mg/kg body weight/day, such as elevated bilirubin, changes in the red blood cell count and elevated absolute and relative liver weights in males and were considered to be adverse. Based on the results of this study, a no-observed-effect-level (NOEL) or no-observed-adverse-effect-level (NOAEL) of the test substance could not be established.

The lowest-observed-adverse-effect-level (LOAEL) for T002675 was 15 mg/kg in rats after 28 days oral (gavage) exposure. The test substance is therefore classified as STOT RE 1 according to the CLP Regulation.