A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

EC Number:

403-410-1

EC Name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Cas Number:

114565-65-0

Molecular formula:

C18 H16 N8 O3 S2 . x C7 H18 N2 . x C4 H11 N O2 . x C3 H9 N O

IUPAC Name:

(3-aminopropyl)diethylamine; 2-(methylamino)ethan-1-ol; 2-[(2-hydroxyethyl)amino]ethan-1-ol; 6-methyl-2-{4-[2-(triaminopyrimidin-5-yl)diazen-1-yl]phenyl}-1,3-benzothiazole-7-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): FAT 11'184/B
- Physical state: powder
- Analytical purity: No data mentioned in the report. However, in an Ames test report (CCR 124312, see IUCLID chapter 7.6.1) using the same batch of test substance the analytical purity was given as 89.6%.
- Lot/batch No.: Op. 1 BD 823/8
- Expiration date of the lot/batch: January, 1993
- Stability under test conditions: test substance in dilution is stable for at least 2 hours

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Designation: KFM-Han. Wistar (outbred, SPF-Quality)
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 8 weeks
- Weight at study initiation: Males, 149 - 175 g; Females, 148 - 174 g
- Housing: in groups of 3 in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba 343, Batch 91/88 rat maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test article was weighed into a glass beaker on a tarred Mettler PK 300 balance and the vehicle was added. The mixture was prepared daily prior to administration using a homogenizer and was kept stable during application with a magnetic stirrer.

REASON FOR DOSE SELECTION
The dose levels used in the present study were selected on the basis of the results of a previous 5 day range finding study (RCC 205582) performed at the same testing facility.

APPLICATION VOLUME
The application volume was 10 ml/kg bw.

VEHICLE
Carboxymethyl cellulose 4% (CMC) in distilled water (Fluka AG, 9470 Buchs, Switzerland)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article / vehicle mixtures were determined during pretest. Intercurrent sampling for analysis were additionally performed during week 3 of testing. Analyses were performed in the RCC Analytical Chemistry Laboratory.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, seven days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1 (control): 10 animals/sex were used and treated during the 28 days testing period; therefrom 5 animals/sex were kept after ending of the treatment period for another 14 day recovery period.
Group 2 (low dose, 50 mg/kg bw/d): 5 animals/sex were used and treated during the 28 days testing period.
Group 3 (mid dose, 200 mg/kg bw/d): 5 animals/sex were used and treated during the 28 days testing period.
Group 4 (high dose, 1000 mg/kg bw/d): 10 animals/sex were used and treated during the 28 days testing period; therefrom 5 animals/sex were kept after ending of the treatment period for another 14 day recovery period.

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL SIGNS
Observations for mortality were recorded daily. Signs of toxicity were assessed daily.

BODY WEIGHTS
The body weight of each animal was recorded weekly during the acclimatization and treatment period.

FOOD CONSUMPTION
The food consumption was recorded once during the acclimatization period and weekly thereafter.

OPHTHALMOSCOPIC EXAMINATIONS
Ophthalmoscopic examinations were performed on all animals at termination of treatment and recovery period, respectively. Ten minutes after the application of a mydriatic solution the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope.

HAEMATOLOGY AND CLINICAL BIOCHEMISTRY
Blood samples for haematology and clinical biochemistry were collected from all animals under light ether anaesthesia at termination of treatment and recovery period, respectively. The animals were fasted for 18 hours before sampling but water was provided. Blood samples were collected from the retro orbital plexus of each animal between 7.00 and 9.30 a.m. to reduce biological variation caused by circadian rhythms.

Haematological parameters
Erythrocyte count (RBC), Haemoglobin (HB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELETS), Reticulocyte count (RETIC), Nucleated erythrocytes (normoblasts, NEN), Heinz bodies (HEINZ-BOD.), Methemoglobin (MET-HB), Total leukocyte count(WBC), Differential leukocyte Count (Diff. WBC Count), Red cell morphology, Coagulation (Thromboplastin time PT and Partial thromboplastin time PTT).

Clinical biochemistry
Glucose, Urea, Creatinine, Total Bilirubin (BILI. T.), Total Cholesterol (CHOLEST. T.), Triglycerides (TRIGL.), Total Phospholipids, Total Aspartate aminotransferase (ASAT/GOT), Alanine aminotransferase (ALAT/GPT), Lactate dehydrogenase (LDH), Creatine kinase (CK), Alkaline phosphatase (ALP), Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein (total).

UNINALYSIS
Urine was collected over an 18-hour period into a specimen vial using a metabolism cage, during which time the animals were deprived of food but allowed access to water ad libitum. Urine sampling was done at termination of treatment and recovery period, respectively.
Following parameters were examined: Volume (18-hour), Specific gravity (SPEC. GRAV.), pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen (UROBILI.), Urine Sediment (SED. MICRO.).

Sacrifice and pathology:

All animals surviving to the end of the test and recovery period were anesthetized by intraperitoneal injection of sodium pentobarbitone and sacrificed for the purpose of necropsy. Moribund animals also were sacrificed.

GROSS PATHOLOGY AND ORGAN WEIGHTS
Following sacrifice, the animals were subjected to gross pathological examination and the weights of following organs were recorded: Adrenals, Brain, Heart, Kidneys, Liver, and Testes.
Samples of the following tissues and organs were collected from all animals and fixed in phosphate buffered neutral 4 % formaldehyde solution: Adrenals; Aorta; Brain; Cecum; Colon; Duodenum; Epididymides; Esophagus; Eyes with optic nerve 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; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary gland, mandibular, sublingual; Seminal vesicles; Sciatic nerve; Skeletal muscle; Skin; Spinal cord, cervical; Spleen; Sternum with marrow; Stomach; Testes; Thymus; Thyroid gland; Tongue; Trachea; Urinary bladder infused with formalin; Uterus with uterine cervix; Gross lesions.

HISTOPATHOLOGY
Organ and tissue samples were processed, embedded, cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. Slides of Adrenals, Heart, Kidneys, Liver, Spleen and Gross lesions (all animals) collected at terminal sacrifice from the animals of the control and high-dose groups were examined. Upon detection of treatment-related changes in the organs of any high-dose animal, histopathological examination of the same organs in all dose groups were performed.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett’s test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-to one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher's exact test was applied. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred.
All animals of group 4 (high-dose) showed orange discoloured faeces between days 5 and 28 of the treatment period. The animals of the recovery group showed the same discoloration between days 1 and 3 of the recovery period (test days 29-31). All recovery (treatment-free) animals had recovered from these findings at day 4 of the treatment-free period (day 32 of test). No other clinical signs were observed in the animals of the control or test article-treated groups.

BODY WEIGHT AND BODY WEIGHT GAIN (for details, see table below)
No statistically significant differences in body weight and body weight gain were observed during treatment and during recovery.

FOOD CONSUMPTION (for details, see table below)
No statistically significant differences which could be related to test article treatment were observed throughout the entire test and recovery period when the food consumption of the control animals was compared to those of the animals of the test article-treated groups.
In fact, a statistically significant decrease in food consumption was observed in female rats of groups 3 and 4 (mid and high dose groups) during pretest, as well as in female rats of group 4 during recovery period. These differences were considered to be incidental and of normal biological variation known for this strain and age.

OPHTHALMOSCOPIC EXAMINATION
Some incidental findings such as e.g. a single case of enophthalmos (male animal) in the control group at test ending, were reported. None of the findings was treatment-related.

HAEMATOLOGY (see more details in table)
Following effects after 4 weeks of treatment were reported for rats of the high dose group (1000 mg/kg bw/d) when compared to controls:
- slightly decreased erythrocyte count (by 9 to 12%) for both sexes;
- slightly decreased haemoglobin concentration (by 12 to 15%) for both sexes;
- slightly decreased haematocrit value (by 12 to 14%) for both sexes.
These findings were reversible at the end of the treatment-free (recovery) period by approximately 5% on average, when compared to controls. The findings reflect a slight anaemic condition and are therefore considered treatment-related. All other differences in the results of the haematological data were considered to be incidental and of normal biological variation for rats of this strain and age (reference values for untreated Wistar/Han. rats were included in the report).

CLINICAL BIOCHEMISTRY (see more details in table)
Following effects were noted after 4 weeks of treatment for rats of the high dose group (1000 mg/kg bw/d), when compared to controls:
- slightly decreased glucose level (by 11%) for males;
- slightly increased urea level (by 26%) for males;
- slightly increased triglyceride level (by 57%) for females;
- slightly decreased phospholipids level (by 12%) for males;
- slightly increased aspartate aminotransferase activity (by 25 to 17%) for both sexes;
- slightly increased calcium level (by 7%) for females;
- slightly increased sodium level (by 3%) for both sexes;
- slightly decreased potassium level (by 6%) for males;
- slightly increased chloride level (by 2 to 3%) for both sexes;
- slightly increased albumin level (by 5 to 11%) for both sexes;
- slightly increased total protein level (by 7%) for females.
The affected parameters were completely revered until termination of the treatment-free (recovery) period. In the absence of demonstrable tissue necrosis, the findings are not considered significant in toxicological terms but may be a reflection of stress induced metabolic adaptation due to the treatment. All other differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation for rats of this strain and age (reference values for untreated Wistar/Han. rats were included in the report).

URINALYSIS
Following effects were noted after 4 weeks of treatment for rats of the high dose group (1000 mg/kg bw/d), when compared to controls:
- slightly decreased overnight urinary output, volume/18 hrs (by 31%) for males (4.8 ml versus 7 ml for control);
- slight proteinuria for females (protein score was 3 versus 1 for control);
- slight bilirubinuria for both sexes (bilirubin score was 1 versus 0 for control) in males and females;
At the end of the treatment-free (recovery) period the findings, with the exception of the bilirubinuria (score still was 1 versus 0), were found to be reversible. The findings may be treatment-related; however, for urine bilirubin false positive reactions are not to be ruled out since findings for the Ictotest reaction (used to confirm bilirubin reactions with N-Multistix) were negative. This is sometimes seen when a compound or metabolite thereof interferes with oxidation tests by giving a masking colour effect. In addition, the bilirubinuria was not supported by an increase in the plasma bilirubin level.
All other differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation (reference values for untreated Wistar/Han. rats were included in the report).

GROSS PATHOLOGY
Several rats from the mid and high dose groups displayed yellowish discoloration of various segments of the gastro-intestinal tract. No further treatment-related.

ORGAN WEIGHTS AND ORGAN WEIGHT RATIOS
At terminal sacrifice after week 4 of the treatment period the male rats of the high dose group (1000 mg/kg bw/d) showed a statistically significant increase in kidney to organ/body and organ/brain weight ratios. Whereas the female rats of both the mid dose (200 mg/kg bw/d) and high dose groups showed statistically significant increase in absolute and relative (body/brain) kidney weights and those of the high dose group in addition increased absolute and relative (body/brain) liver weights.
After 6 weeks, i.e. after the recovery period, the male rats of the high dose group showed statistically increased absolute kidney and organ/body weight ratios, whereas the female rats of the same group showed these effects in absolute and relative organ/body and organ/brain ratios. In addition the female rats showed increased liver to organ/body weight ratios when statistically compared to the findings determined in the animals of the control group.

HISTOPATHOLOGY
The yellowish discoloration of the gastro-intestinal tract noted grossly had in most cases no microscopic correlation. In only one case could dyestuff particles be identified in the lumen of the cecum. A minimal to slight degree of vacuolation of the straight tubules in the kidneys was recorded in all male rats and two female rats of the high dose group, sacrificed at the end of treatment. This change was regarded as an indicator of metabolic adaptation and was not present in any of the rats sacrificed after the two week recovery period.
Other microscopic findings recorded in both sacrifice groups, were spontaneous in nature and are within the normal range of background lesions observed in these ages and this strain of rat.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were seen in the low and mid dose groups.

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: orange discoloured faeces, changes in haematological and clinical chemical parameters, and changes in organ weights (kidney, liver) were reported for the animals of the high dose group.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

DETAILS ON TREATMENT-RELATED CHANGES IN HAEMATOLOGICAL DATA
4 week-treatment period		Females (n=5)							Males (n=5)
Parameters		RBC (T/l)		HB (mol/L)		HCT (l/l)			RBC (T/l)			HB (mol/L)		HCT (l/l)
0 mg/kg bw/d		8.5		10.1		0.49			8.9			10.3		0.5
50 mg/kg bw/d		8.8		10.3		0.50			8.8			10.2		0.49
200 mg/kg bw/d		8.3		9.9		0.48			8.6			10		0.48
1000 mg/kg bw/d		7.4*		8.6*		0.42*			8.0*			9.1*		0.44*
After recovery		Females (n=5)							Males (n=5)
0 mg/kg bw/d		8.2		10.3		0.5			8.8			10.3		0.5
1000 mg/kg bw/d		7.7*		9.5*		0.46*			8.3			9.7*		0.47*
*. Dunnett-Test based on pooled variance significant at 5% (*)
DETAILS ON TREATMENT-RELATED CHANGES IN CLINICAL-BIOCHEMICAL DATA
4 week-treatment period	Females (n=5)
Parameters	Gl (mo/l)		Urea (mmol/l)	TG (mmol/l)	PL (mmol/l)		ASAT (µkat/l)	Ca (mmol/l)		Na (mmol/l)	K (mmol/l)		Cl (mmol/l)	Alb (g/l)	Tot. P g/l
0 mg/kg bw/d	5.34		8.36	0.32	1.71		1.05	2.55		144.1	3.4		109.3	34.4	72
50 mg/kg bw/d	5.61		8.26	0.31	1.81		1.00	2.62		145.1	3.59		109.2	35.2	73.3
200 mg/kg bw/d	5.46		7.97	0.38	1.80		1.02	2.66		146.1*	3.52		110.5	35.7	72.9
1000 mg/kg bw/d	5.71		8.64	0.51*	1.56		1.24*	2.73*		148.4*	3.40		111.9*	38.2*	77.1*
After recovery	Females (n=5)
0 mg/kg bw/d	6.06		8.26	0.34	1.84		1.09	2.46		138.2	3.29		105.6	34	72.8
1000 mg/kg bw/d	6.76*		8.69	0.37	1.58		1.18	2.44		139.2	3.23		105.2	33.9	70.9
4 week-treatment period	Males (n=5)
Parameters	Gl (mo/l)		Urea (mmol/l)	TG (mmol/l)	PL (mmol/l)		ASAT (µkat/l)	Ca (mmol/l)		Na (mmol/l)	K mmol/l)		Cl (mmol/l)	Alb (g/l)	Tot. P g/l
0 mg/kg bw/d	5.61		6.44	0.49	1.45		1.20	2.63		144.9	3.53		108.2	31.5	68.4
50 mg/kg bw/d	5.99		6.50	0.59	1.38		1.19	2.65		144.5	3.62		108.6	31.8	67.8
200 mg/kg bw/d	5.55		7.13	0.46	1.36		1.25	2.62		145.3	3.61		109.2	31.8	67.8
1000 mg/kg bw/d	4.99*		8.09*	0.45	1.28*		1.50*	2.60		149.5*	3.34*		111.3*	32.9*	67.8
After recovery	Males (n=5)
0 mg/kg bw/d	5.6		6.27	0.68	1.41		1.22	2.56		139.2	3.77		105.6	30.9	69.2
1000 mg/kg bw/d	5.74		7.39	0.64	1.57		1.24	2.43*		141	3.46		105.8	31.2	68.4
*. Dunnett-Test based on pooled variance significant at 5% (*)
RBC erythrocyte count; HB haemoglobin; HCT, hematocrit; GL glucose; TG triglycerides; PL phospholipids; ASAT Aspartate aminotransferase; Ca calcium; Na sodium; K potassium; Cl chloride; Alb albumin, Tot P total protein.

DETAILS ON TREATMENT-RELATED CHANGES IN ORGAN WEIGHTS, FEMALES
4 week-treatment period	Females (n=5), mean absolute weights (g)							Females (n=5), mean relative weights (%)
	Body weight	Brain		Liver		Kidney		Liver/body		Liver/brain	Kidney/body			Kidney/brain
0 mg/kg bw/d	193.9	1.82		5.94		1.35		3.07		327.26	0.70			74.34
50 mg/kg bw/d	203.7	1.85		6.49		1.51		3.19		352.3	0.74			81.99
200 mg/kg bw/d	195.8	1.81		6.94		1.61*		3.55		384.45	0.82**			89.29*
1000 mg/kg bw/d	196.1	1.81		7.77**		1.74**		3.95**		428.6**	0.88**			95.87**
After recovery	Females (n=5), mean absolute weights (g)							Females (n=5), mean absolute weights (g)
0 mg/kg bw/d	209.2	1.84		6.41		1.48		3.06	348.96			0.71	80.29
1000 mg/kg bw/d	201.2	1.85		6.91		1.64*		3.44*	373.85			0.82**	88.97*
*.** Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level
DETAILS ON TREATMENT-RELATED CHANGES IN ORGAN WEIGHTS, MALES
4 week-treatment period	Males (n=5), mean absolute weights (g)							Males (n=5), mean relative weights (%)
	Body weight		Brain		Liver		Kidney	Liver/body		Liver/brain	Kidney/body			Kidney/brain
0 mg/kg bw/d	280.4		1.94		8.53		1.96	3.04		438.46	0.70			100.74
50 mg/kg bw/d	266.6		1.92		9.38		2.06	3.54		489	0.77			106.94
200 mg/kg bw/d	269.7		1.89		9.00		2.06	3.33		475.07	0.77			108.64
1000 mg/kg bw/d	259.6		1.90		8.84		2.27	3.40		464.28	0.88**			119.49*
After recovery	Males (n=5), mean absolute weights (g)							Males (n=5), mean relative weights (%)
0 mg/kg bw/d	301.5		1.92		8.78		2.03	2.89	454.78			0.67			105.66
1000 mg/kg bw/d	311.5		1.99		10.00		2.35*	3.21	502.4			0.75*			117.69
*.** Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level