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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 November 2005 - 04 March 2006 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

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

Test material

Constituent 1
Reference substance name:
-
EC Number:
479-300-2
EC Name:
-
Molecular formula:
unspecified
IUPAC Name:
Reaction mass of Bisbenzimidazo[2,1-a:1',2'-b']anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-6,11-dione and Bisbenzimidazo[2,1-a:2',1'-a']anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-10,21-dione
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation: solid, powder
- Particle size distribution (TEM): 30.3 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: spherical
- Surface area of particles: 16.8 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1515 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable
Specific details on test material used for the study:
- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature
- Lot/batch No.: Partie053001
- Expiration date of the lot/batch: 18 November 2020
- CAS No. Cis: 55034-81-6 Trans: 55034-79-2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar Crl : (WI) BR (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: no data
- Housing: 5 animals per sex in Macrolon plastic cages
- Diet (ad libitum): standard pelleted laboratory animal diet (from Altromin (code VRF- 1, Lage, Germany).
- Water (ad libitum): tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range : 19.2 - 22.6°C)
- Humidity (%): 30-70% (actual range : 23 - 94%)
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-U) (Millipore Corporation, Bedford, USA)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose volume: 5 mL/kg bw/day

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at the testing laboratory and on information from the sponsor.
- Purity: (Milli-U) (Millipore Corporation , Bedford, USA).
- Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.
- Storage conditions: At ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance formulations in water (Milli-U) were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 88% to 99% of nominal. The accuracy level of 88% obtained for one group 3 sample was slightly outside the acceptable range of 90 - 110% of nominal. However, since all other accuracy values were within this range and the accuracy results were in line with the procedural recovery results, the overall accuracy for formulations was considered to be acceptable.
Duration of treatment / exposure:
28 days followed by a 14 day recovery period (control and high dose only)
Frequency of treatment:
daily for at least 28 days, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (low and intermediate dose groups)
10 (vehicle control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results of a 5-day range finding study and in consultation with the sponsor, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg bw/day.
- Rationale for selecting satellite groups: There were two satellite (recovery) groups: 0 and 1000 mg/kg bw/day
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for mortality was made at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, detailed clinical observations were made in all animals. Detailed clinical observations were also performed outside the home cage in a standard arena on a weekly basis.

BODY WEIGHT: Yes
- Time schedule for examinations: Treatment period: on days 1, 8, 15, 22 and 28. Recovery period: on days 1, 8 and 14.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes (Weekly)

WATER CONSUMPTION: Yes (but no quantification)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment period.
- Anaesthetic used for blood collection: Yes: iso-flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands)
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, platelets. Clotting Potential: Prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment period.
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos.).

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (approximately 16 hrs) from all animals at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, colour score, Clarity, specific gravity, pH, protein, glucose, ketone, bilirubin, occult blood, leucocytes, nitrite, urobilinogen, sodium, potassium, calcium, sediment (white blood cells (WBC-SED), red blood cells (RBC), casts, epithelial cells, crystals, bacteria.

NEUROBEHAVIOURAL EXAMINATION: Yes (Functional Observations)
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period : 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve [if detectable] and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

Tissues mentioned within brackets were not examined mlcroscopically as there were no signs of toxicity or target organ involvement. Histological examinations were performed on organs and tissues from all Main Group 1 and 4 animals (0 and 1000 mg/kg bw/d), and all gross lesions. All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Heart, Epididymides, Liver, Kidneys, Spleen, Testes, Thymus, Brain.

In addition, a histopathological investigation of liver, kidneys and adrenal glands of the animals of test groups 2 and 3 (100 and 300 mg/kg bw/day) was performed to meet the additional requirements by the Japanese authority (MHLW) (BASF SE, 99P0617/04P001, 30 Apr 2010).
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were observed.
Black faeces observed at 1000 mg/kg bw/day from week 3 of treatment onwards and incidental cases of black staining of parts of the fur were considered to be due to staining properties of the test substance (a black powder). These findings had resolved during the recovery phase. Incidental findings that were noted in single animals during the treatment or recovery phase included alopecia, scabs and a broken tail apex. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in control animals and in males at 300 mg/kg bw/day and females at 100 and 300 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment with the test substance.
The statistically significant lower body weight gain of females at 100 mg/kg bw/day (day 15, 24% compared to 30% in the control) was absent at higher dosages, and was therefore considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was considered to have been unaffected by treatment with the test substance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time (PT) of females at 1000 mg/kg bw/day at the end of the treatment phase was well within the historical control range. Considering the direction (i.e. a decrease) and slight degree of change, this was considered to be of no toxicological significance (17.4 s compared to 18.2 s in the control). The lower mean corpuscular haemoglobin (MCH) level of males at 300 mg/kg bw/day achieving a level of statistical significance occurred in the absence of a treatment-related distribution and was therefore also considered to be of no toxicological significance (1.15 fmol compared to 1.19 fmol in the control).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant deviations in clinical biochemistry parameters were within the historical control range and occurred in the absence of a (clear) dose-related response. Therefore these changes were considered to be without toxicological significance. These changes comprised higher albumin (33.8 and 33.7 g/L as compared to 32.5 g/L in the control) and chloride levels (102, 103, and 102 mmol/L as compared to 101 mmol/L in the control) in females at 100, 300 and/or 1000 mg/kg bw/day, lower aspartate aminotransferase activity levels (ASAT) in males at 100 mg/kg bw/day (71.2 U/L as compared to 85.6 U/L in the control) and higher sodium levels in females at 300 mg/kg bw/day (138.3 mmol/L as compared to 135.9 mmol/L in the control).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in urinary parameters of treated rats.
Any statistically significant changes in urinary parameters of females at 300 mg/kg bw/day were absent at 1000 mg/kg bw/day and were therefore considered to be of no toxicological relevance. These changes comprised a lower urinary volume, a higher specific gravity, and a higher sodium and potassium concentration (absent when corrected for urinary volume).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in organ weights of treated rats.
The lower heart to body weight ratio of males at 100 mg/kg bw/day occurred in the absence of a dose-related distribution (0.319 % as compared to 0.347% in the control) and the mean was well within the historical control range. This change was therefore considered to be of no toxicological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant necropsy findings were observed.
Black contents of the gastro-intestinal tract (or parts thereof, i.e. stomach, caecum and/or colon) in most animals at 1000 mg/kg bw/day and in one male at 300 mg/kg bw/day at the end of the treatment phase were considered to represent test substance (a black powder). These findings had resolved at the end of the recovery phase and occurred in the absence of any correlating histopathological tissue reaction. Therefore, these observations were considered to be of no toxicological relevance. Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, red foci on the kidneys, red discolouration of the thymus, enlarged mandibular lymph node, fluid in the uterus, and tail apex fracture. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. Variations noted in individual motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes of the low or high sensors, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No histopathological abnormalities were observed that were considered to be of toxicological significance.
Black pigment deposits in the lumen or on the mucosal surface of the gastrointestinal tract in 5/5 males and 4/5 females at 1000 mg/kg bw/day occurred in the absence of any histopathological tissue reaction and were absent at the end of the recovery period. Therefore, these observations were considered to be of no toxicological relevance. The range of other microscopic observations recorded in this study was within the normal range of physiological changes and background alterations that may be seen in untreated animals of this age and strain.

In addition, liver, kidney and adrenal glands of all male and female animals of test group 2 and 3 (100 and 300 mg/kg bw/day) were investigated.
Livers of all animals revealed minimal to slight multifocal lymphoid infiltrates, characterized by a randomly scattered distribution of aggregates of lymphoid cells and minimal Kupffer cell granuloma. In addition, two males (1 of test group 2, 1 of test group 3) revealed in the area of lymphoid infiltration/Kupffer cell granuloma minimal single cell necrosis (3-5 hepatocytes) as accompanying finding. In two females of test group 3 an extramedullary hematopoiesis was seen.
One male of test group 2 revealed in the kidney a focal basophilic tubule. Additionally, two females of test group 3 showed a minimal unilateral pyelitis.
Accessory cortical tissue (accessory nodule) was found in the adrenal gland of 2 males and 1 female of test group 3.
All these findings noted were single observations and/or were biologically equally distributed in both test groups. They were all considered to be incidental and spontaneous in nature and not related to treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for the test article of 1000 mg/kg/day was established.
Executive summary:

The repeated dose toxicity of the test article was investigated in guideline and GLP compliant 28-day oral toxicity study by daily gavage in the rat, followed by a 14-day recovery period. Based on the resutls of a 5-day range finding study and in consultation with the sponsor, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg/day. Each group consisted of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery. Formulation analyses were conducted once during treatment to assess accuracy, homogeneity and stability of formulations. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology at the end of treatment; macroscopy at termination; organ weights and histopathology on a selection of tissues. Accuracy, homogeneity and stability over 5 hours of formulations of test substance in Milli-U water were demonstrated by analyses. No toxicologically relevant changes were observed in any of the parameters determined in this study. No evidence for neurotoxicity or immunotoxicity was obtained in this study. NOAEL was therefore determined to be 1000 mg/kg.