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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD 422 compliant study with well characterized material.
Cross-reference
Reason / purpose:
reference to same 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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
: Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Substance type: blue solid
- Physical state: solid
- Purity test date: 2012
- Lot/batch No.: 110075
- Expiration date of the lot/batch: April 12, 2014
- Stability under test conditions: stable
- Storage condition of test material: at room temperature
- Other: The test substance appeared to be homogeneous.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Wistar Rat, Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks
- Weight at study initiation: average 200g (females) and 320g (males)
- Housing: The rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). Pregnant animals and their litters were housed together until day of parturation (PND) 4 (end of lactation).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

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

IN-LIFE DATES: From: 08-18-2011 To: 09-16-2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.

- The administration volume was 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 95-101% of the nominal concentrations.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.
Females: 52 days
Males: 31 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 14-day range-finding study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; a check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. Thereby, the following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals; during the administration period on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning from the retroorbital venous plexus
- Anaesthetic used for blood collection: Yes, isoflurane (Isoba®, Essex GmbH Munich, Germany).
- Animals fasted: Yes
- How many animals: first five parental males and females per group.
- Parameters checked:
• in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameter / Unit / Method / Reference (Operator’s Guide for Advia 120 System)
- Leukocyte count (WBC) / giga/L / cytochemistry coupled with flow cytometry
- Erythrocyte count (RBC) / tera/L / flow cytometric laserlight scattering
- Hemoglobin (HGB) / mmol/L / cyanmethemoglobin method; according to ICSH
- Hematocrit (HCT) / L/L calculation: MCV x erythrocytes
- Mean corpuscular volume (MCV) / fl / RBC/PLT method; mean of RBC volume distribution curve (histogram)
- Mean corpuscular hemoglobin (MCH) / fmol / calculation: hemoglobin, erythrocytes
- Mean corpuscular hemoglobin concentration (MCHC) / mmoL/L calculation: hemoglobin, hematocrit
- Platelet count (PLT) / giga/L / flow cytometric laserlight scattering
- Differential blood count / % and giga/L / cytochemistry coupled with flow cytometry
- Reticulocytes / % / cytochemistry coupled with flow cytometry

• Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Parameter / Unit / Method / Reference
- Prothrombin time (Hepato Quick’s test) (HQT) / seconds / citrated blood with calcium thromboplastin / Fischer, M. and Falkensammer, Ch.,
Klin. Wschr. 86, 577-583 (1974)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning from the retroorbital venous plexus
- Animals fasted: Yes
- How many animals: first five parental males and females per group.
- Parameters checked:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
References (ALT, AST, ALP): Recommendations of the German Society for Clinical Chemistry: "Standardization of methods for determining enzyme activities in biological liquids". J. Clin. Chem. Clin. Biochem. 8, 658-660 (1970); J. Clin. Chem. Clin. Biochem. 9, 464-465 (1971); J. Clin. Chem. Clin. Biochem. 10, 182-192 (1972); Roche working instructions

• Enzyme (systematic name and system number) / Unit / Method, wavelength and measuring temperature
- Alanine aminotransferase (ALT); EC 2.6.1.2. / μkat/L / kinetic UV test, 340 nm; 37°C /
- Aspartate aminotransferase (AST); EC 2.6.1.1. / μkat/L / kinetic UV test, 340 nm; 37°C /
- Alkaline phosphatase (ALP); EC 3.1.3.1. / μkat/L / kinetic color test, 415 nm, 37°C
- γ-Glutamyltransferase (GGT); EC 2.3.2.2. / nkat/L / kinetic color test, 415 nm, 37°C / Szasz, G. et al., J. Clin. Chem. Clin. Biochem. 12, 228 (1974); Roche working instructions

• Blood Chemistry Parameter / Unit / Method / References
- Sodium (NA) / mmol/L / ion selective electrodes (ISE) / Hitachi 917 - working instructions
- Potassium (K) / mmol/L / ion selective electrodes (ISE) / Hitachi 917 - working instructions
- Chloride (CL) / mmol/L / ion selective electrodes (ISE) / Hitachi 917 - working instructions
- Inorganic phosphate (INP) / mmol/L / molybdate reaction Henry, R.J. in: "Clinical Chemistry", Harper and Row Publishers, New York (1974);
Roche working instructions
- Calcium (CA) / mmoL/L / o-cresolphthalein complex without deproteinization / Ray Sarkar, B.C. and Chauhan, U.P.S., Anal. Biochem. 20, 155 (1967); Roche working instructions
- Urea (UREA) / mmoL/L / enzymatic determination with the urease/ glutamate dehydrogenase method / Neumann, U. and Ziegenhorn, J.: XVI,
Nordiska kongressen for klinisk kemi och klinisk fysiologi 1977, Oulu, Finland; Roche working instructions
- Creatinine (CREA) / μmoL/L / kinetic Jaffé method without deproteinization / Bartels, H. et al., Clin. Chim. Acta 37, 193 (1972); Roche working instructions
- Glucose (GLUC) / mmoL/L / hexokinase/glucose-6-phosphate dehydrogenase method / Schmidt, F.H., Klin. Wschr. 39, 1244-1247 (1961); Roche working instructions
- Total bilirubin (TBIL) / μmoL/L / DPD method Wahlefeld, A.W. et al., Scand. J. Clin. Lab. Invest. 29, Suppl. 126 (1972) Abstract 11.12; Roche working instructions
- Total protein (TPROT) / g/L / biuret method Weichselbaum, T.E., Amer. J. Clin. Path. 10, 40 (1946); Roche working instructions
- Albumin (ALB) / g/L bromocresol green method Doumas et al., Clin. Chim. Acta 31, 87 (1971); Roche working instructions
- Globulins (GLOB) / g/L / difference between total protein and albumin
- Triglycerides (TRIG) / mmoL/L / enzymatic color test with lipase esterase/ glycerokinase/ glycerol-3-phosphate oxidase/4-aminophenazone
mod. method by Wahlefeld, A.W., in "Methoden der enzymatischen Analyse" [Methods of enzymatic analysis] (Bergmeyer, H.U., ed.) Vol. II, 3rd ed.,
Verlag Chemie Weinheim, GERMANY, pp. 1878-1882 (1974); Roche working instructions
- Cholesterol (CHOL) / mmoL/L / enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method)
Siedel, J. et al., J. Clin. Chem. Clin. Biochem. 19, 838 (1981); Roche working instructions
- Magnesium (MG) / mmoL/L / xylidylblue method Mann, C.K. and Yoe, J.H., Anal. Chem. 28, 202-205 (1956); Bohnon, C., Clin. Chim. Acta 7, 811-817
(1962)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Parameter / Method / References
- pH / methyl red and bromothymol blue / Test strip book by Roche, Mannheim, GERMANY (1977)
- Protein / tetrabromophenol-phthaleinethylester (TBPE) / Test strip book by Roche, Mannheim, GERMANY (1977)
- Glucose / GOD-POD reaction / Test strip book by Roche, Mannheim, GERMANY (1977)
- Ketones / sodium nitroprusside / Test strip book by Roche, Mannheim, GERMANY (1977)
- Urobilinogen / p-methoxyaniline-diazonium-salt / Test strip book by Roche, Mannheim, GERMANY (1977)
- Bilirubin / 2,5-dichloroaniline diazonium salt / Test strip book by Roche, Mannheim, GERMANY (1977)
- Blood / 2,5-dimethylhexane-2,5-dihydroperoxide, tetramethylbenzidine / Test strip book by Roche, Mannheim, GERMANY (1977)
- Specific gravity / refractometer / Hamilton or Atago operating instructions
- Sediment / microscopy / Hallmann, L., [Clinical Chemistry and Microscopy] 10, ed., 233-246, Georg Thieme, Stuttgart, Germany (1966)
- Color, turbidity / by visual evaluation
- Volume / graduated tubes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: A functional observational battery (FOB) was performed in the first five parental males and females (with litter) per group

- Battery of functions tested:
• passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
• Home cage observations:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings
• Open field observations:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes
• Sensorimotor tests/reflexes:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings
• Motor activity assessment
- carried out in the first five parental males and females (with litter) per group at the end of the administration period.
- measured on the same day as FOB was performed.
- examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany.
The animals did not receive any food or water during the measurements.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
- Weight parameters (determined on all animals):
1. Anesthetized animals
2. Epididymides
3. Testes
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

- Organ / Tissue preservation
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

From the liver, each one slices of the lobus dexter medialis and the lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY: Yes
Organ samples / Methods-Scope of examinations / Test group
1. All gross lesions: Hematoxylin-eosin (H&E), all affected animals per group, all treatment groups
2. Adrenal glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
3. Bone marrow (femur): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
4. Brain: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
5. Cecum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
6. Cervix: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
7. Coagulation glands: Hematoxylin-eosin (H&E), all affected animals per group, control and high dose group
8. Colon: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
9. Duodenum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
10. Epididymides: Hematoxylin-eosin (H&E), all affected animals per group
11. Heart: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
12. Ileum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
13. Jejunum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
14. Kidneys: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
15. Liver: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group; low and mid dose group: embedded in paraplast all animals per group.
16. Lung: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
17. Lymph nodes (mesenteric and axillary lymph nodes): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
18. Ovaries: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
19. Oviducts: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
20. Peyer’s patches Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
21. Prostate: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
22. Rectum: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
23. Sciatic nerve: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
24. Seminal vesicles: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
25. Spinal cord (cervical, thoracic and lumbar cords): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
26. Spleen: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
27. Stomach (forestomach and glandular stomach): Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
28. Testes: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
29. Thymus: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
30. Thyroid glands: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
31. Trachea: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
32. Urinary bladder: Hematoxylin-eosin (H&E), 5 animals per sex per group, control and high dose group
33. Uterus: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
34. Vagina: Hematoxylin-eosin (H&E), control and high dose group, all animals per group
Statistics:
Food consumption, body weight and body weight change: DUNNETT-test (two-sided);
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity; clinical pathology parameters (except for urine color and turbidity), pathology (weight parameters): Non-parametric one-way analysis using KRUSKALWALLIS 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 efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

A blue discoloration of feces was observed on study day 7, 14, 21, 28 and 35 in all animals of the high dose group (1000 mg/kg bw/day).Furthermore, identical discoloration of feces was observed in all females of the mid dose group (300 mg/kg bw/day) on study day 42 and 49. This finding was caused by gastrointestinal passage of the blue test substance. It does not represent an adverse effect.

Applicant's summary and conclusion

Conclusions:
Subacute gavage treatment of rats with doses of 100, 300 and 1000 mg/kg bw/day did not cause treatment-related adverse effects.
Executive summary:

The NOAEL for general, systemic toxicity was 1000 mg/kg bw/day based on the clinical findings, the findings in clinical pathology and histopathology.