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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
616-307-3
EC Number:
616-307-3
Cas Number:
76205-19-1
Molecular formula:
C9 H7 Cl N2 O
IUPAC Name:
616-307-3
Specific details on test material used for the study:
CAS No.: 76205-19-1
Test substance No.: 10/0615-2
Batch identification: L84-174
Purity: 99.6 %
Homogeneity: given (visually)
Stability: stable until 01 Apr 2020
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Housing: rats were housed together (5 animals per cage) in polysulfonate cages
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”,ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C,
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily (administration via diet)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
doses were selected based on the results of a 14-day range finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals as well as for signs of toxicity was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined prior to the start of the administration period. At the end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Following parameters were examined:
Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Following parameters were examined:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined:
pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period
- Battery of functions tested:
Home cage observations: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities
Open field observations: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait abnormalities, Activity/ arousal level, Feces excreted within 2 minutes (number/ appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes
Sensory motor tests/ reflexes:
Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
organ weights: 1. Anesthetized animals, 2. Adrenal glands 3. Brain 4. Epididymides 5. Heart 6. Kidneys 7. Liver 8. Ovaries 9. Prostate 10. Seminal vesicles with coagulating glands 11. Spleen 12. Testes 13. Thymus 14. Thyroid glands 15. Uterus with cervix

HISTOPATHOLOGY: Yes
0 and 1000 mg/kg bw dose groups:
1. All gross lesions 2. Adrenal glands 3. Aorta 4. Bone marrow (femur) 5. Brain 6. Cecum 7. Cervix 8. Coagulating glands 9. Colon 10. Duodenum 11. Epididymides 12. Esophagus 13. Eyes with optic nerve 14. Female mammary gland 15. Femur with knee joint 16. Harderian glands 17. Heart 18. Ileum 19. Jejunum
20. Kidneys 21. Larynx 22. Liver 23. Lung 24. Lymph nodes (mesenteric and axillary lymph nodes) 25. Nose (nasal cavity, level 3) 26. Ovaries 27. Pancreas
28. Parathyroid glands 29. Peyer’s patches 30. Pharynx 31. Pituitary gland

all dose groups: kidneys and spleen
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
Weight parameters:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of
each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Daily drinking water consumption was increased in both sexes of test group 3 (1000 mg/kg bw/d) throughout the observation period, with a measured maximum of +50% in males on study day 77 and +41% in females on study day 49. The increased water consumption in test group 3 (1000 mg/kg bw/d) was assessed as being related to the test substance administration.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the study in male animals of test group 3 (1000 mg/kg bw/d), red blood cell (RBC) counts and hemoglobin values were decreased whereas mean corpuscular volume (MCV), mean hemoglobin content (MCH) and relative reticulocyte counts were increased.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the study in male animals of test group 3 (1000 mg/kg bw/d), total protein and globulin values were decreased whereas triglyceride levels were increased.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of both sexes of test group 3 (1000 mg/kg bw/d), urine volume was higher and the specific gravity of the urine was lower compared to controls. In males, of test groups 2 and 3 (300 and 1000 mg/kg bw/d) more crystals of unknown origin were found in the urine sediment.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Male animal No. 13 of test group 1 (100 mg/kg bw/d) did not walk during observation. Due to the single occurrence and the lack of a dose response-relationship this finding was assessed as being incidental and not test substance-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All macroscopic findings occurred either individually and were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In test group 3 (1000 mg/kg bw/d), the kidneys showed an eosinophilic material occupying unilaterally or bilaterally the lumen of the renal pelvis in 6 of 10 males and 1 of 10 females and urothelial hyperplasia in 7 of 10 males and 3 of 10 females. Although not always simultaneously present, the urothelial hyperplasia most probably represented a reactive response to the presence of the eosinophilic material in the renal pelvis and was regarded as adverse. The composition of the eosinophilic material remains uncertain since special stains for its histochemical origin were negative for glycoproteins, α-2uglobulin or hemosiderin.
Furthermore, medullar mineralization and chronic progressive nephropathy tended to increase in males and females. Since both findings frequently occur spontaneously in animals of this age, they were considered to represent an exacerbated treatment-related effect, which was regarded as non adverse since no clinical chemistry parameters of renal dysfunction were evident.
In the spleen of males of test group 3 (1000 mg/kg bw/d), a minimal increase in the grading of the extramedullary hematopoiesis of erythroid type was noted. This finding was considered to reflect the physiologic regenerative capacity of the spleen to respond to the anemia detected at clinical chemistry. Therefore, the extramedullary erythroid hematopoiesis in the spleen was regarded as treatment-related but not adverse.
In test group 2 (300 mg/kg bw/d), 1 of 10 males showed in the kidneys unilateral eosinophilic material in the renal pelvis associated with minimal hyperplasia of the urothelium, which was considered to be adverse.
In test group 1 (100 mg/kg bw/d), male and female rats showed no treatment-related effects.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
103 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
clinical signs
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
316 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
clinical signs
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion