Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 September 1999 to 4 January 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted : 21st September 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Remarks:

Dark Red Violet

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by KAO Corporation, batch No. 0609RA (lot No. AK0709)
- Expiration date of the lot/batch: May 2001
- Purity test date: March 18, 1999

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:at room temperature, light and humidity protected
- Stability under test conditions: >= 72 hours
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water and stability : >= 72 hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The mixtures of the test article and vahicle were prepared weekly. The test article was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). The mixtures were prepared using a magnetic stirrer. During the daily administration period, homogeneity was maintened using a magnetic stirrer.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanIbm:WIST (SPF)

Details on species / strain selection:

Recognized by international guidelines as a recommended test system

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Biotechnology & Animal Breeding Division
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: male : mean 146 grams / female ; mean 116 grams
- Fasting period before study: not specified
- Housing: Groups of five animals in Makrolon type-4 cages with sterilized standard softwood bedding ('Lignocel') Schill AG (Switzerland)
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 rat maintenance diet (Provimi Kliba AG) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: Seven days under test conditions with an evaluation of health status

DETAILS OF FOOD AND WATER QUALITY: Analysis of contaminants in food and water were performed and reported.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h and at least 8 hours music/light period

IN-LIFE DATES: From: 27 September 1999 To: 4 January 2000

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral ingestion is a possible route of human exposure

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% CMC in bi-distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The mixtures of the test article and vehicle were prepared weekly. The test article was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). The mixtures were prepared using a magnetic stirrer. During the daily administration period, homogeneity was maintened using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): not specified
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity and content of the test article in the dose formulations was determined with the first test article preparations before treatment start. Intercurrent samples for analysis (homogeneity and content) were drawn at weeks 6 and 12. The analysis was performed according a High Performance Liquid Chromatography HPLC method.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose were used

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected according to the results of "Ext. D&C Violet 2 : 14 day oral (gavage) toxicity study in the rat" study report No. RCC Project 736290
- Rationale for animal assignment (if not random): Computer-generated random algorithm

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked : Piloerection, salivation, hunched posture, ataxia, tremore/twitching, prostration, hyperactivity, somnolence, dyspnea, tachypnea, bradypnea

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first test article exposure and once weekly during weeks 1-12

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Acclimatization : on all animals, at week 13 on all animals of groups 1 and 4
- Dose groups that were examined: group 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Bloods samples were collected from all animals early in the working day to reduce biological variation caused by circadian rhythms
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked : Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, reticulocyte fluorescence ratios, nucleated erythrocytes, total leukocyte count, differential leukocyte count, red blood cell morphology, thromboplastin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Bloods samples were collected from all animals early in the working day to reduce biological variation caused by circadian rhythms, after 13 weeks
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked : plasma color, glucose, urea, creatinie, uric acid, bilirubin, lipids, cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma glutamyl transferase, calcium, phosphorus, sodium, potassium, chloride, protein total, protein electrophoresis.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during 18 hours fasting period into a specimen vial. After 13 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked : Volume (18 hours), specific gravity, osmolality, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, nitrite urobilinogen, urine sediment, white blood cells, crystals (triple phosphate)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 13
- Dose groups that were examined:
- Battery of functions tested: grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weighed ; Adrenals, ovaries, brain, spleen, heart, kidneys, testes, thyroid, liver, thymus

HISTOPATHOLOGY: Yes
Organs conserved in neutral phosphate buffered formalin:
Adrenals glands, Aorta, Brain, cerebral cortex, femur, cecum, colon, duodenum, epididymides, esophagus, eyes with optic nerve, heart, ileum, jujenum, kifneys, liver, lungs, lymph nodes, mammary gland area, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, parathyroid gland, tongue, trachea, urinary bladder, uterus, vagina

Statistics:

The following statistical methods were used to analyze the body weights, organ weights and all ratios as well as clinical laboratory data:
If the variables were assumed to follow a normal distribution, the Dunett's test (many to one t-test) based on pooled variance estimate was applied 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 the Dunnett-test when the data could not be assumed to follow a normal distribution.
Student's T-test was applied to locomotor activity and grip strength
Fisher's exact test was applied to the ophtalmoscopy data and macroscopial data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dark respective blue feces were noted in all treated groups with increasing degree depending on time and/or dose level. Discolored feces were noted in some male or female animals treated with 1000 mg/kg/day during week 3 of the study.

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

Description (incidence and severity):

Opacity and pupillary membrane were observed equally in control and test article treated animals and were therefore considered to be not test article related.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

An increased Tombroplastin Time (PT) and Incresed Activated Partial Thromboplastin Time was observed in male rats from the high dose level group (1000 mg/kg/day). Only red blood cells slight increase was observed in the females of the same group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male treated with 300 and 1000 mg/kg/day, in all test article treated females, statistically significant decreased urea were noted. In addition, creatinine levels in males and females treated with 1000 mg/kg/day were statistically significant increased when compared with the controls.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In females with 300 mg/kg/day and 1000 mg/kg/day, respectively, statistically significant decreased locomotor activity was evident after 45 minutes which persisted in females treated with 1000 mg/kg/day until the end of measurement period as well as increases in motor activity in males given 1000 mg active dye/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A slight increase of mean kidney weight was observed on male from the group treated with 100 and 300 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In two males and two females treated with 1000 mg/kg/day, blue discoloration of the mucosal surface of the stomach and / or intestine was observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

haematology

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1 :summary of body weight values

BodyWeight		Group 1 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Week1	Male	184	185	192	187
	SD	16.8	11.7	21	23
	Female	118	116	118	114
	SD	14.3	14.1	15.5	12.4
Week13	Male	376	398	386	390
	SD	34.8	39.4	38.6	27.1
	Female	244	251	242	239
	SD	15.1	18.6	17.2	16.1

 

Table 2 :Summary of Results

HaematologyMeanValue		Group 1 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
PT	Male	12.5	13.1*	12.7	13.0*
ProthrombinTime	Female	12.5	12.6	12.1	12.5
APTT	Male	22.6	23.5	23.1	24.8**
Activated Partial Thromboplastin Time	Female	23.1	22.1	23.1	23.5
Clinical Biochemistry Mean Value
Urea	Male	6.17	5.9	5.31*	5.29*
	Female	7.7	6.49**	6.07**	5.64**
Creatinine	Male	53	55.1	55.9	59.5**
	Female	59.7	59.2	63.7	66.1*
Lipids total	Male	2.93	2.57	2.71	2.55*
	Female	2.57	2.45	2.48	2.78
Cholesterol total	Male	1.98	1.64	1.79	1.7
	Female	1.44	1.37	1.35	1.66
Phospholipids	Male	1.68	1.49	1.56	1.46*
	Female	1.62	1.55	1.61	1.78
Albumin	Male	38.4	37.42	38.24	40.13*
	Female	44.61	41.87	44.79	46.25
Gamma globulin	Male	1.06	1.12	1.31	1.53**
	Female	1.55	1.92*	1.62	1.52
Firbinogen	Male	1.9	2	2.05	1.17**
	Female	1.46	2.01**	1.62	2.02**
Organ weights
Kidneys weight	Male	2.05	2.36**	2.33*	2.2
	SD	0.17	0.24	0.26	0.16
	Female	1.5	1.62	1.6	1.58
	SD	0.13	0.13	0.1	0.17
Spleen	Male	0.729	0.77	0.749	0.732
	SD	0.075	0.09	0.125	0.1
	Female	0.526	0.605*	0.543	0.55
		0.053	0.076	0.091	0.056

 *p<0.05 **p<0.01

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of test substance Jarocol Violet 43 was defined as 300 mg/kg/day on repeated toxicity (gavage) on rats for 90 day period of treatment according to the APTT Activated Partial Thromboplastin increased activity in the high dose level group.

Executive summary:

This GLP compliant study aims the potential toxic effect of the registered item Acide Violet 43 (Jarocol Violet 43) after a 90 days period of treatment on rats by oral gavage. This study was performed following the OECD guideline 408 method for subchronic oral toxicity test on rodent.

Daily oral gavage at 0 (vehicle, 1% aqueous solution of carboxymethylcellulose), 100, 300 or 1000 mg test material substance/kg/day at a dosing volume of 10 ml/kg was performed for 13 weeks. These dose levels were selected on the basis of a previous 14-day oral toxicity study in rats. Evaluations and measurements included mortality, daily cageside observations, weekly body weight, food intake and detailed clinical observations, ophthalmoscopy (on all animals in acclimation period and on control and high dose animals in week 13), as well as functional parameters, haematology, blood clinical chemistry and urinalysis (week 13). At the end of the dosing period, animals were killed and subjected to macroscopic examination, selected organs were weighed, and organs/tissues were preserved. Microscopic examination was performed for specified tissues/organs from control and high dose rats, and for gross anomalies from all animals.

Dark blue feces were observed at all dose levels and were related to the staining properties of the test substance. Statistically significant decreases in motor activity were observed for females given 300 or 1000 mg/kg bw/day as well as increases in motor activity in males given 1000 mg/kg bw/day. In females effects were evident after 45 minutes and persisted in animals treated with 1000 mg/kg bw/day until the end of the observation period. In males effects were observed after 15 minutes only. The dose-dependency of effects in females might give evidence, that effects could be related to the test substance.

Moderate but statistically significant changes in several clinical laboratory parameters including increases in mean clotting time values and in activated partial thromboplastin time in males were reported for the highest dose group. The only findings at necropsy were a blue discolouration of the digestive tract observed for a few animals given 1000 mg/kg/day. No relevant histopathological changes were noted at examination of tissues and organs of treated animals.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of test substance Jarocol Violet 43 was defined as 300 mg of test material/kg/day on repeated toxicity (gavage) on rats for 90 day period of treatment according to the APTT Activated Partial Thromboplastin increased activity in the high dose level group.