Sodium 6,14-diethyl-1,1,1,2,2,18,18,19,19,19-decafluoro-8,12-dioxo-10-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)-4,7,13,16-tetraoxanonadecane-9-sulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 FEB 2012 - 18 JUL 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range finder

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 JAN 2012 - 15 MAR 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

dose range finder for study according to OECD guideline 407

Reason / purpose for cross-reference:

reference to other study

Remarks:

main study

Guideline:

other: dose range finder for study according to OECD Guideline 407

Deviations:

not applicable

Principles of method if other than guideline:

- Principle of test: The study was performed as a dose-range finder for a planned 28-day repeat dose toxicity study in rats.
- Short description of test conditions: Six rats (3 per sex) were treated orally with 1000 mg/kg bw of the test item once daily for 5 consecutive days. Directly before administration the test material was prepared with fully demineralized water as vehicle.
- Parameters analysed / observed: The rats were examined for clinical symptoms and the body weight was recorded. At the end of the study, rats were sacrificed and a macroscopic examination was performed.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Kißlegg
- Age at study initiation: 8 weeks
- Weight at study initiation: The mean initial body weight at the start ofthe study was 220 g (range from 212 to 233 g) for the males and 159 g (range from 151 to 165 g) for the females .
- Housing: The rats were kept individually in type III Makrolon cages with a shelter, placed on mobile racks. Conventional softwood granulate was used as the bedding. The bedding was not changed during the experimental part of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 d

DETAILS OF FOOD AND WATER QUALITY: The drinking water is periodically analyzed according to the German regulations for human drinking water. According to the specifications given by the manufacturer, the diet Provimi Kliba 3433.0, had been checked by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Humidity (%): 48 - 62
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Directly before the administration the test material was prepared with the vehicle. For this purpose, the test material was put into the vehicle and dissolved by stirring the preparation for 1 hour at 40°C. The test material preparations were administered orally by gavage at a dose volume of 10 mL/kg.

Analytical verification of doses or concentrations:

no

Frequency of treatment:

once daily for 5 consecutive days

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Dose selection rationale: The dose level was selected due to the fact, that the test material was tested in a single oral toxicity study at a dose level of 2000 mg/kg without any clinical effects or organ alterations at gross pathological examination. The median lethal dose (LD50) for males and females was higher than 2000 mg/kg bw after an observation period of 15 days.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The behavior and general condition of all rats were checked daily, once immediately after treatment and at least once within 3 hours after treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The behavior and general condition of all rats were checked daily, once immediately after treatment and at least once within 3 hours after treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed predose, on treatment day 1 and then daily at the same time of day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Immediately after the first treatment one male showed salivation. On the consecutive days in all rats salivation was observed immediately after treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight decrease of body weight was observed in one female rat (No. 16) on day 2 und in one further female rat (No. 18) an day 5 of the experimental part. The body weight development of all other rats was inconspicuous throughout the study.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The gross pathological examination revealed no organ alterations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

Immediately after the first treatment one male rat showed salivation. On the consecutive days in all rats salivation was observed immediately after treatment. A slight decrease of body weight was seen in one female rat on day 2 und in one further female rat on day 5 of the experimental part. The body weight development of all other rats was inconspicuous throughout the study. There were no deaths during the course of the study. The gross pathological examination revealed no organ alterations.

Conclusions:

Based on the results of this study 1000 mg/kg bw can be used as the highest dose for the planned 28-day oral repeated dose toxicity study in rats.

Executive summary:

The study was performed as a dose-range finder for a planned 28-day repeat dose toxicity study in rats. Six rats (3 per sex) were treated orally with 1000 mg/kg bw of the test item once daily for 5 consecutive days. Directly before administration the test material was prepared with fully demineralized water as vehicle. The rats were examined for clinical symptoms and the body weight was recorded. At the end of the study, rats were sacrificed and a macroscopic examination was performed. Immediately after the first treatment one male rat showed salivation. On the consecutive days in all rats salivation was observed immediately after treatment. A slight decrease of body weight was seen in one female rat on day 2 und in one further female rat on day 5 of the experimental part. The body weight development of all other rats was inconspicuous throughout the study. There were no deaths during the course of the study. The gross pathological examination revealed no organ alterations. Based on the results of this study 1000 mg/kg bw can be used as the highest dose for the planned 28-day oral repeated dose toxicity study in rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 OCT 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim (20.10.2010)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

sodium 1,5-dioxo-1,5-bis({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy})-3-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)pentane-2-sulfonate

EC Number:

700-541-9

Cas Number:

1472634-24-4

Molecular formula:

C27H34F15NaO12S

IUPAC Name:

sodium 1,5-dioxo-1,5-bis({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy})-3-({[1-(2,2,3,3,3-pentafluoropropoxy)butan-2-yl]oxy}carbonyl)pentane-2-sulfonate

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on species / strain selection:

For the testing of chemicals in repeat-dose 28 day oral toxicity studies in rodents, the preferred species is the rat according to the guidelines mentioned above. Furthermore, the rat is used as a standard rodent species at Merck KGaA’s Non-Clinical Safety.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: about 8 weeks
- Weight at study initiation: male: 219 (205 – 240)g, female: 170 (155 – 185)g
- Fasting period before study: no
- Housing: gang-housed (2 or 3 animals/sex) in type IV Makrolon® cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 22.7 °C
- Humidity (%): 38.3 - 61.3 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 29, recovery group to day day 43

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized water

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): --
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): --
- Purity: ultra pure

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- METHOD OF DETERMINATION
The test item was determined via UPLC-MS/MS on a reversed-phase column in gradient mode. An electro spray tandem mass spectrometer operating in negative ion mode was used as detector. External standards were used for calibration.

- Equipment
Autosampler: Acquity UPLC, WATERS
Binary Solvent Manager: Acquity UPLC, WATERS
Column Manager: Acquity UPLC, WATERS
Detektor: Mass selectiv detector, TQD
Acquity UPLC, WATERS
Software: Empower 2 (Build no: 2154), WATERS

- Additional equipment
Direct displacement pipettes, Microman, GILSON
Piston stroke pipettes, Finnpipette, THERMO SCIENTIFIC

- Reagents
Methanol, gradient grade, VWR
HPLC water, for HPLC, VWR
Acetonitrile, super gradient, VWR

- Standard
The test item was used as external standard.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 times a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group 1 control (0 mg/kg bw): 20 (10m/10f)
Group 2 (100 mg/kg bw): 10 (5m/5f)
Group 3 (300 mg/kg bw): 10 (5m/5f)
Group 4 (1000 mg/kg bw): 20 (10m/10f)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A single oral dose of 2000 mg/kg was tolerated in male and female Wistar rats, the LD50 was considered to be higher than 2000 mg/kg bw. In a 5-day dose range finding oral toxicity study in rats a dose of 1000 mg/kg bw/d was tolerated in male and female Wistar rats.
The high dose for the 4-week repeat dose toxicity study was set according to OECD 407 recommendations and the results of the 5-day study to 1000 mg/kg bw/d. Moderate systemic toxic effects were expected at this dose level. 100 and 300 mg/kg bw/d were chosen to be able to characterize dose-dependencies of effects.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: according to OECD TG 407, recovery groups are required
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (working days twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily (working days twice daily)

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment and then once a week

FOOD CONSUMPTION : yes
- Time schedule for examinations: once a week

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: twice a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in weeks 5 and 7
- Anaesthetic used for blood collection: Yes (inhalation anesthesia)
- Animals fasted: Yes
- How many animals: 10 (5m/5f) from control and high dose group
- Parameters checked below were examined.
Parameter Unit Abbreviation Instrument/ Method
Red blood cells (erythrocytes) /pL RBC (a)
Hemoglobin g/dL HGB (a)
Hematocrit % HCT (a)
Mean cell volume fL MCV (a)
Mean hemoglobin content pg MCH (a)
Mean hemoglobin concentration g/dL MCHC (a)
Platelets /nL PLT (a)
Reticulocytes % RET% (a)
Absolute number of reticulocytes /nL RET (a)
White blood cells (leukocytes) /nL WBC (a)
Absolute number of neutrophilic granulocytes /nL NEUT (a, b)
Absolute number of lymphocytes /nL LYM (a, b)
Absolute number of eosinophilic granulocytes /nL EOS (a, b)
Absolute number of basophilic granulocytes /nL BASO (a, b)
Absolute number of monocytes /nL MONO (a, b)
Absolute number of large unstained cells /nL LUC (a, b)
Neutrophilic granulocytes % NEUT% (a, b)
Lymphocytes % LYM% (a, b)
Eosinophilic granulocytes % EOS% (a, b)
Basophilic granulocytes % BASO% (a, b)
Monocytes % MONO% (a, b)
Large unstained cells % LUC% (a, b)
Prothrombin time sec PT sec (c)
Prothrombin time % PT % (c)
Partial thromboplastin time sec PTT (c)

a) ADVIA 120, Siemens Medical Solutions GmbH (Bad Nauheim, Germany)
b) Visual differentiation by a microscope, ZEISS (Oberkochen, Germany)
c) Coasys Plus – Thrombolyzer (Diasys-Greiner, Germany)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in weeks 5 and 7
- Anaesthetic used for blood collection: Yes (inhalation anesthesia)
- Animals fasted: Yes
- How many animals: 10 (5m/5f) from control and high dose group
- Parameters checked below were examined.

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

a) ADVIA 1650, Autoanalyzer, Siemens Medical Solutions Diagnostics GmbH, (Bad Nauheim, Germany)

URINALYSIS: Yes
- Time schedule for collection of urine: 18 hours before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked below were examined.
Parameter in Urine Abbreviation Method Instrument
pH value - pH Reflectometry (b)
Protein PRO Reflectometry (b)
Glucose GLU Reflectometry (b)
Bilirubin BIL Reflectometry (b)
Blood BLO Reflectometry (b)
Urobilinogen URO Reflectometry (b)
Ketone KET Reflectometry (b)
Sediment SED Visual (c)
Specific gravity SG Refractometry (d)
Urine weight Urine weight Measured by WNT

b) Clinitek ADVANTUS, 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)


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: day 7 and day 28
- Dose groups that were examined: 5m and 5f per group
- Battery of functions tested:
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,
urination (number of fecal boluses, feces consistency, number of urine pools, urine stain size),
hindlimb foot splay,
forelimb,
hindlimb grip strengths,
internal body temperature,
catalepsies,
posture,
vocalization,
convulsions,
stereotypy,
any abnormalities.

MOTOR ACTIVITY
- Time schedule for examinations: day 28
- Dose groups that were examined: the numerical first 5 males and 5 females per group

On day 28, 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 and 7 beams on the z-axis). These parameters are called “counts” and given in numbers. The system was also calculating the “on-time” (minutes), “off-time” (minutes), the “total distance” the animal was moving (meter), “rearing” (number), and “rearing time” (minutes). The data were transferred manually into the LIM-System. Assignment of the rats to the individual measurements followed a randomization.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Terminal body weight (after exsanguination)
Heart
Liver
Kidneys (together)
Spleen
Thymus
Testes (together)
Prostate
Uterus
Ovaries (together)
Adrenals (together after fixation)
Thyroids with Parathyroids (together after fixation)
Brain (after fixation)
Epididymides (together)
Seminal vesicles

HISTOPATHOLOGY: Yes

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

Statistics:

The following standard statistical methods have been applied for data processing to compare dose groups with control group:
- Multiple two-sided Dunnett-Test to evaluate absolute body weight, body weight gain, body temperature, food and water consumption, and organ weights (relative and absolute) and Standard t-test to compare these parameters in the recovery period between the control (group 1) and high dose (group 4).
- Wilcoxon rank sum test to evaluate clinico-chemical parameters and the hematological parameters and correction was done according to Bonferroni-Holm. Wilcoxon rank sum test only to compare in the recovery period between the control (group 1) and high dose (group 4).
- Individual values, mean values and standard deviations to evaluate motor activity data
- Non-parametric 2-sided Kruskal-Wallis test, followed by the Wilcoxon-test rank sum test to evaluate all numerical parameters of the Functional Observational Battery (FOB). Additionally, mean values, number of animals (N), minimum, maximum, median, and the frequencies of scores were calculated for each parameter.

Evaluation was done using the LIM system (except the frequencies of scores).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs showed dose-dependent salivation together with rooting in the bedding material. These observations in rodents usually indicate a bad taste of the test item preparation rather than a toxicological effect. In one group 3 (300 mg/kg) male and two group 4 (1000 mg/kg) males incomplete closure of eyes, prone position, and decreased spontaneous activity were observed on single occasions. Three group 4 (1000 mg/kg) males showed respiratory sounds.

Mortality:

no mortality observed

Description (incidence):

All rats survived the treatment or recovery period until their scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption did not show any treatment-related changes during the treatment and recovery period.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption was statistically significantly increased in group 4 (1000 mg/kg) males during the treatment period. Also group 3 (300 mg/kg) males showed a slight increase of water consumption, which was statistically significant from day 17-21. A slight increase of water consumption was seen in group 3 (300 mg/kg) and 4 (1000 mg/kg) females during the treatment period, statistical significance was noted from day 3-7 (group 4 only), then on days 7-10, 17-21, and day 21-28 in both groups. It is assumed that the increased water consumption could have been caused by the dose-dependent increase of salivation. No correlation with changes in urinalysis, serum clinical chemistry parameters, or histopathological changes in the urinary tract were observed.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

4-week oral administration of the test item at doses of 100, 300 or 1000 mg/kg to Wistar Crl:WI (Han) rats did not show any alterations in hematological and coagulation parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

4-week oral administration of the test item at doses of 100, 300 or 1000 mg/kg to Wistar Crl:WI (Han) rats did not show any alterations clinical chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

4-week oral administration of the test item at doses of 100, 300 or 1000 mg/kg to Wistar Crl:WI (Han) rats did not show any alterations in urine parameters.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In group 4 (1000 mg/kg) females, a trend towards decreased activity was noted - a slight increase of off-time, together with a slight decrease of on-time, decrease of total distance, rearing time and rearing number, mainly caused by 1/5 animals tested in this group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The determination of body and organ weights in main kill animals revealed significantly increased absolute and relative liver weights in group 4 (1000 mg/kg) males and increased relative liver weights in group 3 (300 mg/kg) males when compared with controls. No relevant changes were found in females. After a two week recovery period no statistically significant differences between mean absolute and relative liver weights of group 4 recovery males were found when compared with the controls. No relevant body and organ weight changes were found in females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related lesions were found in main kill and recovery animals in the organs examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology revealed treatment-related findings in the liver of two high dose (1000 mg/kg) males. Both animals showed a minimal to mild centrilobular hepatocellular hypertrophy. Following a two week period of recovery the treatment-related lesions in the liver of males were fully reversible. Females from all dose groups as well as males from the mid (300 mg/kg) and low (100 mg/kg) dose groups revealed no lesions that could be related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

NEUROBEHAVIOUR
In the functional observational battery no treatment-related changes in autonomous, neuromuscular, sensomotoric, central nervous system including internal body temperature were observed. Measurement of motor activity did not reveal any clear treatment-related effects. In group 4 (1000 mg/kg) females, a trend towards decreased activity was noted - a slight increase of off-time, together with a slight decrease of on-time, decrease of total distance, rearing time and rearing number, mainly caused by 1/5 animals tested in this group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: findings at 300 and 1000 mg/kg bw in water consumption

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study the NOAEL (no observed adverse effect level) was established at 1000 mg/kg bw/d, the NOEL (no observed effect level) was considered to be 100 mg/kg bw/d.

Executive summary:

Study design

This GLP study was performed according to OECD TG 407, adopted October 03, 2008. 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 bw/d. A similarly constituted control group received the vehicle, deionized water, 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 and mid dose groups consisted of 5 male and 5 female rats each. At the end of the treatment period 10 (5 males and 5 females) rats per group were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period. The rats were gang-housed under conventional conditions.

The inlife investigations have been performed as described below:

Observations/Measurements	Time schedule
Appearance and behavior	Daily
Mortality	Daily
Motor activity	Day 28
Functional observational battery	Predose (day 0), day 7, day 28
Body weight	Once a week
Food consumption	Once a week
Water consumption	Twice a week
Hematology	Week 5 + 7
Clinical chemistry	Week 5 + 7
Urinalysis	Week 5 + 7

All rats were subjected to macroscopic and histopathological examinations. Selected organs were weighed from each surviving rat at the end of the treatment or recovery period.

Results

Formulation analysis revealed that the dose groups received the anticipated concentrations and no test material was detected in the control formulations.

All rats survived the treatment or recovery period until their scheduled necropsy.

Clinical signs showed dose-dependent salivation together with rooting in the bedding material. These observations in rodents usually indicate a bad taste of the test item preparation rather than a toxicological effect. In one group 3 (300 mg/kg) male and two group 4 (1000 mg/kg) males incomplete closure of eyes, prone position, and decreased spontaneous activity were observed on single occasions. Three group 4 (1000 mg/kg) males showed respiratory sounds. Body weight, body weight gain and food consumption did not show any treatment-related changes during the treatment and recovery period. Water consumption was statistically significantly increased in group 4 (1000 mg/kg) males during the treatment period. Also group 3 (300 mg/kg) males showed a slight increase of water consumption, which was statistically significant from day 17-21. A slight increase of water consumption was seen in group 3 (300 mg/kg) and 4 (1000 mg/kg) females during the treatment period, statistical significance was noted from day 3-7 (group 4 only), then on days 7-10, 17-21, and day 21-28 in both groups. It is assumed that the increased water consumption could have been caused by the dose-dependent increase of salivation. No correlation with changes in urinalysis, serum clinical chemistry parameters, or histopathological changes in the urinary tract were observed. In the functional observational battery no treatment-related changes in autonomous, neuromuscular, sensomotoric, central nervous system including internal body temperature were observed. Measurement of motor activity did not reveal any clear treatment-related effects. In group 4 (1000 mg/kg) females, a trend towards decreased activity was noted - a slight increase of off-time, together with a slight decrease of on-time, decrease of total distance, rearing time and rearing number, mainly caused by 1/5 animals (no. 52) tested in this group. Analysis of hematology, coagulation, clinical chemistry, or urinalysis parameter at the end of treatment and recovery period did not result in any findings that could be attributed to the treatment with the test material. At necropsy, no treatment-related lesions were found in main kill and recovery animals in the organs examined. The determination of body and organ weights in main kill animals revealed significantly increased absolute and relative liver weights in group 4 (1000 mg/kg) males and increased relative liver weights in group 3 (300 mg/kg) males when compared with controls. No relevant changes were found in females. After a two week recovery period no statistically significant differences between mean absolute and relative liver weights of group 4 recovery males were found when compared with the controls. No relevant body and organ weight changes were found in females. Histopathology revealed treatment-related findings in the liver of two high dose (1000 mg/kg) males. Both animals showed a minimal to mild centrilobular hepatocellular hypertrophy. Following a two week period of recovery the treatment-related lesions in the liver of males were fully reversible. Females from all dose groups as well as males from the mid (300 mg/kg) and low (100 mg/kg) dose groups revealed no lesions that could be related to treatment.

Conclusions

Daily oral administration of 100, 300 or 1000 mg/kg of the test material to rats for 4 weeks was well tolerated. The few clinical signs observed at 300 mg/kg and 1000 mg/kg as well as the increased water consumption in both dose groups were not correlated with clinico-chemical or histopathological changes and are not considered as adverse. In histopathology hepatocellular hypertrophy in the liver of two males was observed at 1000 mg/kg and it is considered to be an adaptive and non-adverse effect. Full recovery of this lesion was present after a two week treatment-free period.

Under the conditions of the present study the NOAEL (no observed adverse effect level) was established at 1000 mg/kg, the NOEL (no observed effect level) was considered to be 100 mg/kg.