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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:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride
EC Number:
916-466-1
IUPAC Name:
Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride
Details on test material:
Identification : C059 (Basic Blue 99)
Batch number : 74/75
3-[(Bromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (main) : 62.8a/a%
3-[(Dibromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (E) : 11.8a/a% 3-[(Bromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (F) : 8.4a/a%
3-[(Bromo-5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (J) : 4.1a/a% 3-[(8-Amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (A) : 1.7a/a% 3-[(Sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (B) : 1.8a/a%
3-[(Dibromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (G): 2.1a/a%
3-[(Sulpho-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (O) :1.0a/a%
3-[(Bromo-sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (N) : 0.7a/a%
Tribromo-8-amino-5-hydroxy-1,4-naphthoquinone (L) : 2.6a/a%
Chloride ion : 20.2%
Sulphate ion : 0.5%
Acetate ion : 2.0%
Zinc ion : 6.6%
Water : 7.9%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
Dose Formulation
Dose levels were in terms of the test item as supplied. The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added. The dose formulations were prepared daily and during administration homogeneity was maintained by stirring.
Frequency of formulation : Daily
Stability of formulations : Max 4 hours at 15-25 deg C
Analysis of dose formulations : Concentration and homogeneity of the dose formulations were determined in samples taken during weeks -1, 5, 9 and 13. Stability (4 hours at 15-25 deg C, 7 days at ~4 deg C) was determined from week -1 samples. The test item was used as the analytical standard. Analyses were performed by HPLC.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
60 mg/kg bw/day
Basis:
actual ingested
Dose / conc.:
240 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
240 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Ten males and ten females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Method : Oral, by gavage
Rationale for method : Oral uptake is the possible route for human exposure
Target dose levels : 0, 10, 60, 240mg/kg bw/day
Dose volume/frequency : 10mL/kg body weight/daily administration
Duration of acclimatisation period : 7 days
Duration of treatment period : 91 days
Duration of recovery period : 28 days
Rationale for dose level selection : The dose level selection was based on results of a previously performed oral 90 day non GLP study in rats (20, 60, 180/360 mg/kg bw/day). Effects were seen as dose related pigmentation in stomach, small intestine and adrenals and slight retardation of body weight gain in high dose animals. The NOAEL was 60mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:
Observations
Viability/mortality : Twice daily
Clinical signs : At least once daily during acclimatisation, treatment and recovery periods
Detailed clinical observations : Weekly (during acclimatisation, treatment weeks 1-12 and recovery weeks 14-17)
Food consumption and body weights : Once weekly during acclimatisation, treatment and recovery periods
Water consumption : At weeks 1, 5, 12 (24 hour periods)
Ophthalmoscopy : Once during acclimatisation (allocation), at treatment end (week 13), at recovery end (week 17)
Functional observational battery : Relevant parameters from a modified Irwin screening test were performed on all rats at week 13
Haematology : Blood samples were taken from all animals after week 13 and from the recovery animals after week 17. The animals were fasted for approximately 18 hours before blood sampling but water was provided. The following parameters were determined (erthrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, platelet count, reticulocyte count, reticulocyte maturity index, metheoglobin, heinz bodies (after 13 weeks only), total leukocyte count, differential leukocyte count, coagulation (thromboplastin time, activated partial thromboplastin time)
Clinical biochemistry : The following parameters were examined (glucose, urea, creatinine, bilirubin, cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl transferase, sodium, potassium, chloride, calcium, phosphorus inorganic, protein, albumin, globulin)
Urinalysis : The following parameters were examined (volume, specific gravity, colour, appearance, pH, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes, leukocytes, sediment)
Sacrifice and pathology:
Necropsy
All animals were weighed before necropsy and descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetised by intraperitoneal injection and sacrificed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed (adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, harderian gland, heart, ileum,
jejunum, kidneys, larynx, lacrimal gland, liver, lungs, lymph nodes, mandibular, mammary gland area, nasal cavity, ovaries, oviducts, pancreas, pituitary gland, prostate gland, rectum, salivary gland, parotid, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord spleen, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, gross lesions). The following organs were examined (adrenal glands, aorta, bone marrow (femur), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, harderian gland, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mandibular, mammary gland area, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary gland, parotid, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina, gross lesions).

Organ weights
The following organ weights were recorded on the scheduled dates of necropsy and organ to terminal body weight ratios as well as organ to brain weight ratios determined (brain, heart, liver, thymus, kidneys, adrenals, spleen, testes, epididymides, ovaries, uterus).

Histotechnique
All organ and tissue samples to be examined were processed and cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin.

Histopathology
Histological examination was performed on all study plan organs and tissues from control animals and animals exposed to 60 and 240 mg/kg bw/day of the main study, as well as on all gross lesions from all animals of main and recovery groups. Because of suspected test item related findings noted in the animals treated at 60 and 240 mg/kg bw/day, the adrenal glands, kidneys, lung were examined in the male animals of the 10 mg/kg bw/day dose group and of the recovery groups dosed at 0, 60, 240 mg/kg bw/day.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Deeply blue stained urine observed in animals of 60 and 240 mg/kg bw/day dose groups and coloured faeces excreted by animals of all treatment groups were considered due to the intense blue colour of the test item or metabolites of the test item.
Mortality:
no mortality observed
Description (incidence):
No deaths were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight development was not affected by the treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The overall mean food and water intakes did not show relevant differences between controls and treated groups.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The overall mean food and water intakes did not show relevant differences between controls and treated groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There was no evidence of eye toxicity at the week 13 and 17 examinations.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Minimally increased MCHC (240mg/kg bw/day dose group), slightly increased Met-Hb (240mg/kg bw/day dose group males and females), a slight shift in the reticulocyte maturity index (higher H reti counts, 60mg/kg bw/day and 240mg/kg bw/day females), a minimally increased basophil count (Baso 240mg/kg bw/day males), a minimally higher count for large unstained cells (Luc, 240mg/kg bw/day males). Except for the highe Luc count (240mg/kg bw/day males), all parameters returned to normal ranges at recovery end.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower glucose levels (60mg/kg bw/day and 240mg/kg bw/day dose group males), slightly lower urea concentration (240mg/kg bw/day dose group males), slightly lower creatinine concentration (240mg/kg bw/day dose group males and females), slightly to markedly lower bilirubin concentration (60mg/kg bw/day and 240mg/kg bw/day dose group males, 240mg/kg bw/day dose group females), minimally higher sodium ion concentration (60mg/kg bw/day and 240mg/kg bw/day dose group males), slightly higher chloride ion concentration (60mg/kg bw/day and 240mg/kg bw/day dose group males, 240mg/kg bw/day dose group females), slightly higher calcium ion (240mg/kg bw/day dose group males), slightly higher phosphorus levels (60mg/kg bw/day and 240mg/kg bw/day dose group males) and slightly lower phosphorus levels (60mg/kg bw/day and 240mg/kg bw/day dose group females).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Slightly higher pH (240mg/kg bw/day dose group females) and protein,bilirubin concentrations (240mg/kg bw/day dose group animals), slightly higher ketone concentration (240mg/kg bw/day dose group males), higher counts for erythrocytes (240mg/kg bw/day females) and for leukocytes (240mg/kg bw/day dose group animals). All affected urine parameters returned to normal ranges at recovery end.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Values for grip strength and locomotor activity revealed no treatment related changes.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The analysis of absolute and relative organ weights revealed no differences indicating an effect of the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy at treatment end revealed bluish discolouration in the stomach of 5/10 males of the mid dose group and in 8/10 males and 2/10 females of the high dose group.
Details on results:
Mortality
No deaths were observed.

Clinical Observations
Deeply blue stained urine observed in animals of the 60 mg/kg bw/day and 240 mg/kg bw/day dose groups and coloured feaces excreted by animals of all treatment groups were considered due to the intense blue colour of the test item or metabolites of the test item.

Functional Observational Battery
Values for grip strength and locomotor activity revealed no treatment related changes.

Food And Water Consumption
The overall mean food and water intakes did not show relevant differences between controls and treated groups.

Body Weight
The mean body weight development was not affected by the treatment.

Ophthalmoscopy
There was no evidence of eye toxicity at the week 13 and 17 examinations.

Clinical Laboratory Investigations
Hematology : Minimally increased MCHC (4 males of the 240 mg/kg bw/day group), slightly increased Met-Hb (males and females of the 240 mg/kg bw/day group), a slight shift in the reticulocyte maturity index (higher H Reti counts, females of the 60 mg/kg bw/day and 240 mg/kg bw/day groups), a minimally increased basophil count (Baso, males of the 240 mg/kg bw/day group), a minimally higher count for large unstained cells (Luc, males of the 240 mg/kg bw/day group). Except for the higher Luc count (males of the 240 mg/kg bw/day group), all parameters returned to normal ranges at recovery end.

Clinical biochemistry : Slightly lower glucose (Gluc) levels (males of 60 mg/kg bw/day and 240 mg/kg bw/day groups), slightly lowe urea concentration (males of 240 mg/kg bw/day group), slightly lower creatinine concentration (males and females of 240 mg/kg bw/day group), slightly to markedly lower bilirubin concentration (males of 60 mg/kg bw/day and 240 mg/kg bw/day groups, females of 240 mg/kg bw/day group), minimally higher sodium ion concentration (males of 60 mg/kg bw/day and 240 mg/kg bw/day groups), slightly higher chloride ion concentration (males of 60 mg/kg bw/day and 240 mg/kg bw/day groups, females of 240 mg/kg bw/day), slightly higher calcium ion (males of 240 mg/kg bw/day group), slightly higher phosphorus levels (males of 60 mg/kg bw/day and 240 mg/kg bw/day groups) and slightly lower phosphorus levels (females of 60 mg/kg bw/day and 240 mg/kg bw/day groups). Except for the higher phosphorus level in males of 240 mg/kg bw/day group, all parameters returned to normal ranges at recovery end.

Urinalysis : Slightly higher pH (females of 240 mg/kg bw/day group) and protein and bilirubin concentrations (animals of 240 mg/kg bw/day group), slightly higher ketone concentration (males of 240 mg/kg bw/day group), higher counts for erythrocytes (females of 240 mg/kg bw/day group) and for leukocytes (animals of 240 mg/kg bw/day group). All affected urine parameters returned to normal ranges at recovery end.

Organ Weights
The analysis of absolute and relative organ weights revealed no differences indicating an effect of the test item.

Macroscopic Findings
Necropsy at treatment end revealed bluish discolouration in the stomach of 5/10 males of 60 mg/kg bw/day dose group and in 8/10 males and 2/10 females of the 240 mg/kg bw/day dose group. Following the recovery period, the liver had accentuated lobular pattern in all 240 mg/kg bw/day dose group males.

Microscopic Findings
Microscopic examination revealed increased incidence and average grading of tubular hyaline droplets in the kidneys of males at 240 mg/kg bw/day. Such hyaline droplets are known to represent alpha-2 microglobulins, specific for male rats and to be of no toxicological relevance. In the liver, there were no morphological changes corroborating the macroscopic finding of accentuated lobular pattern.

Effect levels

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: morphological changes to organs

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, a NOEL could not be established. In the absence of morphological changes to organs and tissue the minimal and reversible effects on clinical laboratory parameters, the NOAEL is defined at 60 mg/kg bw/day for males and females.
Executive summary:

In this subchronic toxicity study, daily administration (gavage) of Basic Blue 99 to Wistar rats at doses of 10, 60 and 240 mg/kg bw/day for 13 weeks followed by a 4 week treatment free recovery period (control, mid and high dose only) resulted in no early mortality, no effects on food and water consumption and body weight development. There was no evidence of eye toxicity. Effects recorded upon daily and weekly clinical observations were restricted to stained urine and faeces associated with the intence blue colour of the test item. Measurements performed during the functional observational battery revealed no relevant changes to grip strength and motor activity. Clinical laboratory investigations revealed only minimal to slight changes to few red blood cell parameters (MCHC, Met-Hb, H Reti), granulocytes (Baso) and large unstained cells (Luc). In addition, the lower concentrations of glucose, urea and creatinine are considered of poor diagnostic relevance and the minimal to slight extent of their impairment is not considered of an adverse nature. In the absence of any signs of anemia, the depressed bilirubin levels diagnosed in males and females remain of equivocal toxicological relevance. A minimal disturbance to the electrolyte/water balance in mid and high dose males was substantiated by higher sodium, calcium and chloride concentrations. In females, there was only a minimal and reversible increase of the chloride concentration. Changes to the phosphate concentrations noted in mid and high dose animals (males : increase, females : decrease) were of opposite direction and poor dose relationship. This finding persisting to recovery end in high dose males, is considered of equivocal toxicological relevance. At necropsy, the absolute and relative mean organ weights were not affected and macroscopic changes were restricted to bluish discolouration in the stomach of mid and high dose males and high dose females. At recovery end, accentuated lobular pattern was noted in all high dose males. Microscopically, there were no changes of toxicological relevance to the morphology of tissues and organs. Under the conditions of this study, a NOEL could not be established. In the absence of morphological changes to organs and tissue the minimal and reversible effects on clinical laboratory parameters, the NOAEL is defined at 60 mg/kg bw/day for males and females.