Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-12-2012 to 19-01-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, (2000).
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
482-160-5
EC Name:
-
Cas Number:
130786-09-3
Molecular formula:
C12H13N
IUPAC Name:
(2Z)-2-phenylhex-2-enenitrile
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: Refrigerator (2 to 8°C), in the dark under nitrogen
- Other: colourless

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: males 177.8 – 198.1 g and females 138.3 – 156.2 g; individuals were randomly allocated to treatment groups using a randomization procedure.
- Fasting period before study: None
- Housing: Macrolon cages with sawdust bedding, changed at appropriate intervals; group housed (5 per group) by sex. During locomotor investigations were housed individually.
- Diet (e.g. ad libitum): pelleted rodent diet, certified (recognised supplier), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: pelleted rodent diet, certified (recognised supplier) – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 to 70 %
- Air changes (per hr): 10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 01-12-2012 to 19-01-2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. The test item was prepared at the appropriate concentrations as a suspension in Polyethylene Glycol. The test item was weighed into a tared glass container on a suitable precision balance and the vehicle, PEG 300, was added to give the appropriate final concentration of the test item in the suspension. 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. The test item was found to be stable in application formulations when kept 2 hours at room temperature or 7 days at refrigerator due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Polyethylene Glycol was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable in vehicle.
- Concentration in vehicle: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110% : actual 93.2% to 110.4%. Thus, the required content limit of ±20% with reference to the nominal concentration was met). No test item was detected in the Group 1 (vehicle) formulations. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative-vehicle, untreated group) and all treatment groups with applicable test item concentrations per group.
- Other: Dose-formulations were analysed during the study and were reported as within ± 20% applied limits.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110% : actual 93.2% to 110.4%. Thus, the required content limit of ±20% with reference to the nominal concentration was met). No test item was detected in the Group 1 (vehicle) formulations. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
- The analysis consisted of GC-FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). These were then subjected to analysis by GC-FID analysis using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report). The test item samples were subject to a known dilution (details available within the full study report) to place the end samples in the calibration range.
- Mean concentrations of dose-formulations analysed during the study were within ± 20% applied limits confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Low – Group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Intermediate – Group
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
High – Group
No. of animals per sex per dose:
5 per sex per dose (5 male / 5 female)
Additional 5 animals per sex and group (control and high dose group only) were treated for 28 days and then allowed a 14-day treatment-free recovery period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 5-day sighting study (Report number attached to and cited in the full study report). Dose levels in the 5-day sighting test were: Group 1: 200 mg/kg bw/day (in Polyethylene Glycol), Group 2: 600 mg/kg bw/day and Group 3: 1000 mg/kg bw/day. In the 5-day range finder (administered consecutively, for 5-days) within male/female (two individuals per sex group). On day 5 of treatment, one female at 1000 mg/kg bw/day showed mortality, and the other female at that dose level was humanely euthanised. No abnormal clinical signs were observed in males at 1000 mg/kg bw/day, whereas females showed signs from day 3 (sedation) and weakened condition (day 5), ataxia, reduced body temperature, ptosis and ruffled fur. Males and females showed no abnormal signs at 600 mg/kg bw/day. Food consumption was reduced in males and females at 1000 mg/kg bw/day. Females showed a dose response for food consumption. Body weights for 200 and 600 mg/kg bw/day compared with controls. At 1000 mg/kg bw/day all males/females showed reduced body weights compared to controls. Elevated liver weight ratio was observed at all dose levels and particularly at 600 mg/kg bw/day. Other effects were considered adaptive effects. Macroscopic observations were not considered test item related. At 1000 mg/kg bw/day thymic discoloration was noted in one male and female. At up to 600 mg/kg bw/day no abnormal macroscopic findings were noted. Basis: other: nominal in vehicle (Polyethylene glycol).
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: Yes. 14-day recovery satellite groups were assigned. Specifically, an additional 5 animals per sex and group (control and high dose group only) were treated for 28 days and then allowed a 14-day treatment-free recovery period.
- Section schedule rationale: Random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily. During treatment, twice daily on days 1 to 2; three times daily on day 3, twice daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily. During treatment, twice daily on days 1 to 2; three times daily on day 3, twice daily thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes.
- Body weight gain % was determined.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals and/or end of recovery period (week 6) for satellite recovery groups.
- Anaesthetic used for blood collection: Yes. isoflurane (recognised supplier)
- Animals fasted: Yes. Overnight (18 hours).
- How many animals: All animals
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), Leukocyte count, total, Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count, Methemoglobin Heinz bodies, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals and/or end of recovery period (week 6) for satellite recovery groups.
- Animals fasted: Yes. Overnight (18 hours).
- How many animals: All animals
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, total Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein, total Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes.
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals and/or end of recovery period (week 6) for satellite recovery groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Overnight (18 hours).
- Parameters checked: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Kidneys, Liver, Ovaries

HISTOPATHOLOGY: Yes
- Organs and tissues examined and/or preserved in neutral buffered 4% formalin (where appropriate) except for eyes with optic nerve and harderian gland which were fixed in Davidson's solution or epididymides and testes which
were fixed in Bouin’s solution : Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain - including section of medulla/pons, cerebral and cerebellar cortex, Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes w/optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart including auricles, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland, exorbital, Liver, Lungs, filled w/formalin at necropsy, Lymph nodes - mesenteric, mandibular, Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pharynx, Pituitary gland, Prostate gland incl. coagulating glands, 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, if possible), Tongue, Trachea, Ureter, Urinary bladder, filled w/formalin at necropsy, Uterus, Vagina, All gross lesions,

The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals, where applicable: all gross lesions from all groups
- where any test item related morphologic changes were detected in organs in high-dose the same organs were subject to examination in low and mid dose groups.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was 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 could not 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):
No adverse clinical signs of toxicity were noted during the observation period.

Salivation was noted in all dose groups (grade 1 or 2) in males/females.

Dyspnea (grade 1) was sporadically noted in group 3 (200 mg/kg bw/day) and group 4 (600 mg/kg bw/day).

Salivation and dyspnea was not noted during recovery period and therefore considered to be reversible. Salivation was noted immediately after the oral application of the test item and generally no longer seen during second daily clinical observation.

Dyspnea was not accompanied by any specific macroscopical or microscopical findings at necropsy.

Due to reversibility, no findings at the functional observational battery and no correlated macroscopic or microscopic findings at necropsy, the findings of salivation and dyspnea were considered to be test item-related, but of non-adverse character.
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality.

One female (no 40) of control group 1 died on day 24 of treatment period. This was considered as potentially related to aspiration of the vehicle during gavage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg bw/day there were decreased body weights noted in males/females.
At 60 and 200 mg/kg bw/day and there were no test item related changes in mean absolute and relative body weights noted.

Absolute and relative body weights recovered to normal during recovery period. The findings were therefore considered to be generally reversible. The findings of decreased absolute and relative body weights in high dose animals towards the end of treatment period or at the beginning of recovery period were therefore considered to be test item-related, but not adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg bw/day there were increased absolute food consumption in males and females during treatment period and/or increased relative food consumption in males and females during treatment and recocery period.
At 60 and 200 mg/kg bw/day there were no test item related changes iin the mean absolute and relative food consumption noted.

The 600 mg/kg bw/day findings correlated with slightly decreased mean absolute and relative body weights in high dose animals during treatment period or at the beginning of recovery period in a compensating manner. The findings of increased absolute and relative food consumption was therefore considered to be not adverse.
Food efficiency:
not examined
Description (incidence and severity):
See body weight and weight changes section.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the haematological parameters examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: The following statistically significant findings were noted after 4 weeks:
• Decreased glucose in males and females (both p<0.01).
• Elevated urea in males (p<0.05).
• Decreased creatinine in males (p<0.05) and females (p<0.01).
• Elevated total bilirubin in males and females (both p<0.01).
• Elevated alanine aminotransferase (ALAT) in males (p<0.01) and females (p<0.05).
• Elevated sodium in males (p<0.01).
• Elevated potassium in males and females (both p<0.01).
• Decreased chloride in females (p<0.01).
• Elevated calcium in males (p<0.05).
• Elevated phoshorus in males (p<0.01).
• Elevated protein in males and females (both p<0.01).
Elevated albumin in males (p<0.01).
• Elevated cholesterine and triglycerides in females (p<0.01).

The following statistically significant findings were noted in animals at 600 mg/kg/day (group 4) after recovery period (at week 6):
• Elevated triglycerides in males (p<0.05).
• Decreased lactate dehydrogenase (LDH) in males (p<0.05).
• Decreased alkaline phosphatase (ALP) in males (p<0.05).
• Elevated protein in males (p<0.05).

Since findings of changes in triglycerides, LDH and ALP were single in incidence, not noted after treatment period and values stayed well within ranges of historical reference data the findings were considered to be not test item-related. Slightly elevated protein after recovery period in males treated with 600 mg/kg/day correlated with slightly elevated hematocrit, but was not accompanied by changes in albumin and globulin fractions of the blood. This finding was therefore considered to be of no toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: The following statistically significant findings were noted after 4 weeks:
Decreased glucose in males and females (both p<0.01).
• Elevated urea in males (p<0.05).
• Decreased creatinine in males (p<0.05) and females (p<0.01).
• Elevated total bilirubin in males and females (both p<0.01).
• Elevated alanine aminotransferase (ALAT) in males (p<0.01) and females (p<0.05).
• Elevated sodium in males (p<0.01).
• Elevated potassium in males and females (both p<0.01).
• Decreased chloride in females (p<0.01).
• Elevated calcium in males (p<0.05).
• Elevated phoshorus in males (p<0.01).
• Elevated protein in males and females (both p<0.01).
• Elevated albumin in males (p<0.01).
• Elevated cholesterine and triglycerides in females (p<0.01).

Due to distribution in the high dose group and additional findings in hematology parameters, body weights and histopathology, these findings were considered to be test item-related. Elevated bilirubin and ALAT was considered to be possibly connected to changes of centrilobular hypertrophy noted in the liver in histopathology which was not noted after recovery period anymore.

Due to reversibility after recovery period, the changes in biochemistry parameters in animals treated with 600 mg/kg/day (group 4) were considered to be test item-related, but non-adverse.

The following statistically significant findings were noted in animals at 600 mg/kg/day (group 4) after recovery period (at week 6):
• Elevated triglycerides in males (p<0.05).
• Decreased lactate dehydrogenase (LDH) in males (p<0.05).
• Decreased alkaline phosphatase (ALP) in males (p<0.05).
• Elevated protein in males (p<0.05).

Since findings of changes in triglycerides, LDH and ALP were single in incidence, not noted after treatment period and values stayed well within ranges of historical reference data findings were considered to be not test item-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the urinalysis parameters examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: The following statistically significant findings were noted after 4 weeks:
Elevated relative density in males group 4 (p<0.05) with values above ranges of historical reference data.
• Elevated protein in males group 4 (p<0.01) with values at the upper range of historical reference data.
• Elevated ketones in males and females group 4 (p<0.01 and p<0.05).
• Elevated ketones in males group 3 (p<0.05).
• Elevated urobilinogen in males and females group 4 (both p<0.01) with values markedly above ranges of historical reference data.
• Elevated urobilinogen in males and females group 3 (p<0.01 and p<0.05) with values markedly above ranges of historical reference data.
• Elevated leucocytes in males and females group 4 (p<0.01).
• Elevated leucocytes in males and females group 3 (p<0.05).
• Elevated leucocytes in males and females group 2 (no statistical significance and p<0.01).
• Amber color and turbid appearance was noted in individual data generally in all males and females group 4.
• Yellow or amber color and turbid appearance was noted in individual data in some males and females group 3.

No findings were noted in urinalysis parameters after recovery period.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the behavioural parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related non-adverse findings were noted in animals treated with 60, 200 and 600 mg/kg/day (group 2, 3 and 4).

Changes in absolute and relative liver and kidney weights showed a dose-dependent distribution, were noted in both sexes and correlated to findings of centrilobular hepatocellular hypertrophy and tubular epithelial hypertrophy in the kidneys in histopathology. These findings were therefore considered to be test item-related. Due to reversibility of organ weights and microscopical changes after recovery period, these changes were considered to be not adverse.

The following statistically significant changes in organ weights were noted in animals at 600 mg/kg/day (group 4) after recovery period (week 6):
• Decreased body weights in males and females (p<0.05 and p<0.01).
• Increased heart and adrenals to body weight ratios in males (p<0.05 and p<0.01).
• Increased liver and kidneys to body weight ratios in females (p<0.05).

Decreased body weights were considered to be test item-related. Since relative body weights (body weight gain) were increased during the recovery period in the in-life determination of body weights, this finding was considered to be not adverse.

Due to low incidence and no changes in absolute and/or organ to brain weight ratios, the findings of increased heart and adrenals to body weight ratios were considered to be a secondary effect caused by slightly reduced body weights and not test item-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related macroscopic findings were noted in animals treated with 60 mg/kg/day (group 2) and in females treated with 200 and 600 mg/kg/day (groups 3 and 4).
Test item-related non-adverse findings were noted in males treated with 200 and 600 mg/kg/day (groups 3 and 4).

The following macroscopical findings were noted at necropsy (week 4):
• Enlarged liver: One male (1/5) at 200 mg/kg/day (group 3) and one male (1/5) at 600 mg/kg/day (group 4)
• Alopecia, cheek region: One female (1/5) at 600 mg/kg/day (group 4)

Alopecia in one high dose female was considered to be an incidental finding
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related non-adverse findings were noted in animals treated with 200 and 600 mg/kg/day (group 3 and 4).

At 600 mg/kg bw/day dose level: The following statistically significant findings were noted after 4 weeks:
Centrilobular hypertrophy in the liver, grade 1 or 2 (minimal, slight): 5/5males and 5/5 females with mean severity 1.4 and 1.6, respectively
• Increased hematopoiesis in the spleen, grade 1, 2 or 3 (minimal, slight, moderate): 5/5 males and 3/5 females with mean severity of 1.8 and 1.0, respectively
• Tubular hypertrophy in the kidneys, grade 1 or 2 (minimal, slight): 5/5 males and 5/5 females with mean severity of 1.8 and 1.4, respectively

The findings were not present in the 600 mg/kg bw/day recovery group.

At 200 mg/kg bw/day dose level: The following statistically significant findings were noted after 4 weeks:
• Centrilobular hypertrophy in the liver, grade 1 (minimal): 5/5males with mean severity 1.0
• Increased hematopoiesis in the spleen, grade 1 or 2 (minimal, slight): 5/5 males and 3/5 females with mean severity of 1.2 and 1.0, respectively
• Tubular hypertrophy in the kidneys, grade 1 or 2 (minimal, slight): 1/5 males with mean severity of 1.0

Findings in the liver, spleen and kidneys were noted in a dose-dependent distribution and generally correlated with findings in hematology, biochemistry, urinalysis, organ weights and macroscopical findings. Therefore, these findings were considered to be test item-related. Due to complete reversibility during recovery period, these findings were considered to be not adverse.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected.
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Remarks:
All effects were determined to be non-adverse up to the highest dose level

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 600 mg/kg body weight per day.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7 and US EPA OPPTS 870.3050 guidelines under GLP conditions. Following a previously conducted 5-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day oral gavage study in Wistar: HannRcc: WIST(SPF) rats. Three groups, each comprising five male and five female rats, received test item at doses of 60, 200 or 600 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene Glycol). Further satellite groups of 0 (control) and 600 mg/kg bw/day were similarly treated for 28 days and then allowed a 14 day recovery period. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 2 hours at room temperature or 7 days under refrigeration. Formulation analyses confirmed that formulations of test substance in polyethylene glycol were prepared accurately and homogenously and were stable. Application formulations investigated during the study were found to comprise test item in the range of 93.2% to 110.4%. Clinical signs, food consumption and body weights were recorded periodically during acclimatization, treatment and recovery period. During week 4 of the treatment period, a functional observational battery including measurement of grip strength and locomotor activity was performed. No test item-related mortalities were noted during treatment or recovery period. No adverse findings were noted during daily clinical observations and no findings were noted during weekly detailed behavioural observations at any dose level tested. There were not changes in mean grip strength or mean locomotor activity values for test item treated animals compared to controls. Increased absolute and relative food consumption was noted in animals at 600 mg/kg/day (group 4) during treatment or recovery period. These findings were considered to correlate with slightly decreased mean absolute and relative body weights in high dose animals during treatment period or at the beginning of recovery period in a compensating manner. Test item-related decreased body weights were noted in both sexes at 600 mg/kg/day. Absolute and relative body weights caught up during recovery period. The findings were therefore considered to be not adverse. Hematological changes in the red blood cell parameters like MCV, MCHC, HDW and reticulocytes were considered to be indicative for an anemic, most likely hemolytic, occurrence during the treatment period. Elevated relative and absolute reticulocytes along with elevated H-reticulocytes were considered to clearly reflect a compensatory response. Changes in hematology and biochemistry parameters were considered to be test item-related, but of non-adverse character and reversible. Test item-related non-adverse findings noted in animals treated with 60, 200 and 600 mg/kg/day were elevated absolute and relative liver weights (males and females group 2, 3 and 4), elevated absolute kidney weights (males and females group 2, 3 and 4) and elevated relative kidney weights (males and females group 3 and 4, females group 2). Slightly increased liver and kidneys to body weight ratios in females after recovery period were markedly reduced compared to values of high dose females at week 4 and also comparable to control values at week 4 and therefore considered to be in general reversible. Test item-related non-adverse findings noted in males treated with 200 and 600 mg/kg/day were enlarged liver (1/5 males group 3 and 1/5 males group 4). Since this finding was not noted after recovery period, it was considered to be not adverse. Test item-related non-adverse findings noted in animals treated with 200 and 600 mg/kg/day were centrilobular hypertrophy in the liver (minimal or slight), increased hematopoiesis in the spleen (minimal, slight or moderate), and tubular hypertrophy in the kidneys (minimal or slight). Incidence and severity grades showed a dose-dependent distribution, but were fully reversible during recovery period. It was considered that all effects observed were non-adverse, adaptive and/or were fully reversible at up to 600 mg/kg bw/day. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 600 mg/kg/day for males/females.