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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-06-26 To N/A
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 408.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
481-170-7
EC Name:
-
Cas Number:
502453-61-4
Molecular formula:
Hill formula: C23H30BrN3O2 CAS formula: C23H30N3O2.Br
IUPAC Name:
dimethyl(3-{[4-(methylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}propyl)propylazanium bromide
Constituent 2
Reference substance name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
IUPAC Name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
Details on test material:
- Name of test material (as cited in study report): B119 HC Blue 16
- Molecular formula (if other than submission substance): C23H30N3O2Br
- Molecular weight (if other than submission substance): 460.42
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance):N/A
- Substance type: Active
- Physical state: Dark blue powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd.
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 133.3-153.9 g (mean 143.4 g), Females: 112.0-131.1 g (mean 121.6 g)
- Fasting period before study: N/A
- Housing: Makrolon type-4cages with wire mesh tops and standardized softwook bedding
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 rat maintenance diet, ad libitum
- Water (e.g. ad libitum):Community tap-warter from Itingen, ad libitum
- Acclimation period: 7 days


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


IN-LIFE DATES: From: 2006-06-26 To: 2006-10-30

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Bidistilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (15-25°C). Homogeneity of the test material in the vehicle was maintained during the daily administration period useing a magnetic stirrer.


DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): N/A, oral gavage
- Storage temperature of formulation: (15-25°C)


VEHICLE
- Justification for use and choice of vehicle (if other than water): Bidistilled water
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity, and stability (after 4 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during weeks 5, 9, and 13 of the treatment. The analyses were performed by RCC Ltd. (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon results of a non-GLP dose-range finding study (provided by the Sponsor) in which the B119 HC Blue 16 was administered by oral gavage.
- Rationale for animal assignment (if not random): computer-generated random algorithm
- Rationale for selecting satellite groups: 5/sex/dose (0 and 300 mg/kg bw/day) treated for 91 days under the same conditions and kept for an additional 28 day treatment-free recovery period and sacrificed thereafter.
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): N/A
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: mortality and viability (recorded twice daily), general cageside observations made daily, once before commencement of administration; once daily during treatment and recovery period.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly


BODY WEIGHT: Yes
- Time schedule for examinations: weekly during pretest, treatment and recovery and before necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY: N/A
- 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 data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and weeks 13 and 17
- Dose groups that were examined: Pretest in all animals, at week 13 in control and high dose animals and at week 17 in control and high dose animals.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: After week 13 (all animals) and 17 (only the satellite group)
- Anaesthetic used for blood collection: Yes (Isoflurance)
- Animals fasted: Yes, 18 hours (but allowed access to water ad libitum)
- How many animals: All animals
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscularhemoglobin, Mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Heinz bodies, Total leukocyte count, Differential leukocyte count, Coagulation of Thromboplastin time and activated partial thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After week 13 (all animals) and 17 (only the satellite group)
- Animals fasted: Yes, 18 hours (but allowed access to water ad libitum)
- How many animals: all animals
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Phospholipids, Aspartate aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein (total), Albumin, Globulin, Albumin/Globulin ratio


URINALYSIS: Yes
- Time schedule for collection of urine: After week 13 (all animals) and 17 (only the satellite group)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 hours (but allowed access to water ad libitum)
- Parameters checked: Volume (18 hours), Specific gravity (relative density), Osmolality, Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weeks 13 and 17
- Dose groups that were examined: All animals
- Battery of functions tested: Grip strength, Locomotor activity


OTHER: Blood samples for the determination of serum insulin levels were drawn by heart puncture from all rats prior to their respective exsanguinations and necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; All animals surviving to scheduled necropsy were weighed and necropsied. The following organs and tissues were collected: Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 section planes), Cecum, Colon, Duodenum, Epididymides, Esophagus, Exorbital lacrimal glands, Eyes with optic nerve, Harderian gland, Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and mandibular), Mammary gland area, Nasal cavity (turbinates), Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic and lumbar), Spleen, Stomach, Testes (with parathroid gland), Tongue, Trachea, Urinary bladder, Uterus, Vagina, and Gross lesions.

HISTOPATHOLOGY: Yes; Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 section planes), Cecum, Colon, Duodenum, Epididymides, Esophagus, Exorbital lacrimal glands, Eyes with optic nerve, Harderian gland, Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and mandibular), Mammary gland area, Nasal cavity (turbinates), Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic and lumbar), Spleen, Stomach, Testes (with parathroid gland), Tongue, Trachea, Urinary bladder, Uterus, Vagina, and Gross lesions.
Other examinations:
ABSOLUTE AND RELATIVE ORGAN WEIGHTS: The following organs weights were recorded on the scheduled dates of necropsy: Brain, Heart, Liver, Thyroids with parathyroids, Thymus, Kidneys, Adrenals, Uterus, Spleen, Testes, Epididymides, Ovaries. The organ-to-terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory data, organs weights and ratios, ophthalmoscopic data and macroscopic data: The Dunnetts-test (many to one t-test) based on a pooled variance estimate were 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 Dunnett-test when the data cannot be assumed to follow a normal distribution. And the Fisher’s exact test was also used.

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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals except for two survived until scheduled necropsy. Two females treated with 300 mg/kg bw/day died. One animal died after oral gavage on day 34 and showed bluish discoloration on the animal (subcutis, organs) and distended bluish lungs at necropsy and the other animal was found dead on day 76 of treatment. This animal showed bluish discoloration of the whole animal (subcutis, organs), incompletely collapsed lungs and beginning autolysis at necropsy.
General cageside observations: At 300 mg/kg bw/day, blue discoloration of the feces was noted in both sexes from day 4 of treatment until necropsy or until day 2 of recovery (satellite group). At 100 mg/kg bw/day, blue discoloration of the feces was noted in both sexes from day 17 of treatment until necropsy.
Detailed clinical observations: At 100 and 300 mg/kg bw/day, blue feces were noted in animals from weeks 5 onwards.

BODY WEIGHT AND WEIGHT GAIN
In males treated with 100 and 300 mg/kg bw/day, mean body weight gains were marginally lower from week 5 of treatment on compared to controls, although a statistically significant increase of body weight gain was noted on day 8 (p< 0.01). Males treated with 30 mg/kg bw/day showed lower mean weight gains from week 5 until week 8 of treatment period compared to controls, but higher mean body weight gains from week 9 onwards. In females treated with 30, 100 and 300 mg/kg bw/day, mean body weight gains were higher throughout the acclimation and treatment periods when compared to controls. The difference attained statistical significance (p< 0.05) during weeks 6, 7, 10, 11 and 12 at 100 mg/kg bw/day.
The above-mentioned minor differences in the mean body weights and mean body weight gain values of the test material treated rats were not considered to be related to the treatment with the test material.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In males treated with 300 mg/kg bw/day, the mean daily food consumption was significantly decreased during days 85-91 (p< 0.05), whereas in females of this dose group significantly increased during days 1-8, 8-15, and 78-85 (p<0.05). In females treated with 100 mg/kg bw/day, the mean daily food consumption was significantly increased during days 1-8 (p<0.05). In males treated with 30 mg/kg bw/day, the mean daily food consumption was significantly increased during days 50-57 (p< 0.05) and days 85-91 (p< 0.010).
These minor differences in food consumption were not dose related and considered to be within the range of biological variation and without relation to an effect of the test material.

FOOD EFFICIENCY
N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
N/A

OPHTHALMOSCOPIC EXAMINATION
N/A

HAEMATOLOGY
When compared with the control values from week 13, the following statistically significant differences were noted at 300 mg/kg bw/day after week 13 (end of treatment): males showed elevated hematocrit (p< 0.01) decreased mean corpuscular hemoglobin concentration (p<0.05), decreased hemoglobin distribution width (p<0.01), decreased absolute and relative neutrophils (p<0.05 for absolute only) and statistically reduced methemoglobin level (p<0.05); Females showed decreased hemoglobin distribution width (p< 0.05), elevated mean absolute and relative basophils (both p< 0.085).
These findings represent biological variations, below the physiological range, did not correlate with any histological changes and were therefore not considered to be adverse changes related to the treatment with the test material. Consequently, all observed differences were considered to be of no toxicological relevance.

CLINICAL CHEMISTRY
When compared with the control values from week 13, the following statistically significant differences were noted at 300 mg/kg bw/day after week 13 (end of treatment): changes in electrolytes-elevated sodium (p<0.01 in males, p<0.05 in females), elevated potassium in males (p<0.01) and females (p<0.05), elevated chloride in both sexes (p<0.01), elevated calcium in both sexes (p<0.05 in males and p<0.01 in females); elevated lactate dehydrogenase in females (p< 0.05), elevated creatine kinease in females(p<0.05), decreased urea (males, p<0.05), and decreased alanine aminotransferase activity (males, p< 0.01); reduced triglyceride (p<0.01) and phospholipid (p<0.05) level in males.
When compared with the control values from week 13, the following statistically significant differences were noted at 100 mg/kg bw/day after week 13 (end of treatment): changes in electrolytes-increased sodium in males (p<0.05), elevated chloride in both sexes (p<0.01); decreased triglycerides (males, p<0.01).
When compared with the control values from week 13, the following statistically significant differences were noted at 30 mg/kg bw/day after week 13 (end of treatment): changes in electrolytes-elevated chloride in both sexes (p<0.05 in males, p<0.01 in females), increased potassium ( p< 0.05 in females); reduced aspartate aminotransferase activity (males, p<.05).
The following differences were noted in rats previously treated with 300 mg/kg bw/day after week 17 (end of recovery): elevated alkaline phosphatase (p<0.01 in females); decreased potassium (p<0.05 in males); and decreased calcium, protein, and globulin (p<0.05 in females).

It was noted that these findings represented biological variations, remained within the physiological range and did not correlate with any histopathological changes. Therefore, they were not considered to be adverse changes related to the treatment with the test substance.

URINALYSIS
Increased mean leukocytes count, noted in females treated with 100 and 300 mg/kg bw/day, exceeded the range of the historical control date. Evaluation of the individual data revealed two females with highly elevated leukocytes, whereas the remaining females compared favorably with those of the controls. Elevated leukocyte counts were also noted in three males treated with 300 mg/kg bw/day, but the mean value for these males did not attain statistical significance. After week 17 (end of recovery) all observed changes were reversible.
The discoloration of the urine is related to the staining properties of the test material, or its possible metabolites. The elevation of the leukocyte count may represent more inflammatory processes in the urinary tract and the vaginal, or preputial region, rather than a response to toxic effect. In addition these elevations of the leukocyte count were without concomitant findings in biochemical markers of kidney damage, without histological changes or occurred n isolated animals, therefore, these findings were considered to be incidental changes of no toxicological relevance and also not to represent any substantial infective inflammatory process.

NEUROBEHAVIOUR
Grip strength: The mean hindlimb grip strength was significantly decreased in females of all treatment groups (p<0.01 at 300 mg/kg bw/day, p<0.05 at 100 and 30 mg/kg bw/day).
After recovery week (week 17), the mean hindlimb grip strength values of animals treated with 300 mg/kg bw/day were significantly increased in males and females (both p< 0.01). The mean forelimb grip strength values in these males, however, were significantly decreased (p< 0.01).
Since corresponding clincial signs were not noted and similar differences were not seen for the forelimb grip strength values, the differences associated with hindlimb grip strength were considered to be unrelated to the treatment and with the test material.
Locomotor activity: Females treated with 300 mg/kg bw/day had significantly increased locomotor activity during 0-10 minutes (p<0.01) when compared with the controls. In males treated with 100 mg/kg bw/day, a decrease in mean total locomotor activity at the 0-10 minutes (p<0.01) and 0-60 (p<0.01) overall time intervals at week 13 was noted. The changes noted were not supported by a dose-response relationship and there were consdired to be unrelated to treatment with the test substance.


ORGAN WEIGHTS
In females treated with 300 mg/kg bw/day, increased heart to body weight ratio attained statistical significance (p< 0.05) after 17 weeks. The findings were considered to be biological variation and not test substance related.

GROSS PATHOLOGY
No test substance-related macroscopic findings of toxicological relevance were reported

HISTOPATHOLOGY: NON-NEOPLASTIC
No test substance-related microscopic findings of toxicological relevance were reported.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
N/A

HISTORICAL CONTROL DATA (if applicable)
N/A

OTHER FINDINGS
INSULIN LEVELS: No differences in insulin levels for all treated groups, male and female rats, as compared to the control group were observed. Consequently, there were also no differences between the recovered male and female animals and the control animals.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Excretory discoloration
Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No significant adverse effects were observed at the highest dose tested.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Applicant's summary and conclusion

Conclusions:
Based on the results on this sub-chronic study in rats, the NOEL of B119 HC Blue 16 was established as 30 mg/kg bw/day based upon excretory discoloration, where as the NOAEL was considered to be greater than 300 mg/kd bw/day.
Executive summary:

The test material B119 HC Blue 16 was administered daily by oral gavage to SPE-bred Winstar rats of both sexes at dose levels of 30, 100, and 300 mg/kg bw/day for a period of 91/92 days (13 weeks). The vehicle was bidistilled water. The dose volume in control and test material treated groups was 10 ml/kg bw/day.

The groups comprised 10 animals per sex, which were sacrifice after 91 and 92 days of treatment. Five additional rats per sex were treated for 91 days at 0 and 300 mg/kg bw/day under the same conditions. These latter rats were kept for an additional 28-days treatment-free recovery period and sacrificed thereafter. Clinical signs were recorded daily, whereas outside cage observations, food consumption and bodyweights were recorded weekly during acclimation, treatment and recovery periods. Ophthalmospic examinations were performed during the acclimation period and at the end of treatment and recovery periods. The Observations for the functional observational battery, locomotor activity, and grip strength were performed during week 13 (all animals groups) and 17 (satellite group of 5/sex/dose).

At the end of the dosing and the treatment-free periods, blood samples were withdrawn for hematology, plasma chemistry and serum insulin analyses. Urine samples were collected for urinalysis. All animals were sacrificed, necropsied and examined post mortem. Histological examinations were performed on organs and tissue from all control and test material-treated animals, and all gross lesions from all animals.

Dose formulations were prepared at the test facility and several representative analytical samples were collected and dispatched to the test site internally. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the peak.

The content of B119 HC Blue in all samples was found to be within the accepted range of ± 20% of the nominal content and was found to be homogeneously distributed in the vehicle of the dose formulations. The application formulations were found to be stable for at least 4 hours and 7 days when kept under storage conditions.

No animals died as a result of systemic toxicity, although two females treated with 300 mg/kg bw/day died prior to the scheduled necropsy. One was considered likely to be a gavage error although no confirming macro-or microscopic findings were found (a rupture of the esophagus was not evident, although bluish staining of some organs were noted). No findings were seen in the second female from which a definite cause of death could not be established.

No test material-related changes of toxicological relevance were noted in the following parameters: daily or weekly observations (weeks 1-2, 4-12); functional observational battery (week13), ophthalomoscopic examinations mean daily food consumption or urinalysis, mean serum insulin levels, organ weight and ratios, macroscopical and microscopical findings.

Findings of passive excretory discoloration noted in males and females treated at 300 mg/kg bw/day and at 100 mg/kg bw/day. No test material related changes were noted in the 30 mg/kg bw/day groups.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected.

Based on the results on this sub-chronic study in rats, the NOEL of B119 HC Blue 16 was established as 30 mg/kg bw/day based upon excretory discoloration, where as the NOAEL was considered to be greater than 300 mg/kd bw/day.