Trisodium 5-[[5-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulphonatophenyl]azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-3-methanesulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-740-3

EC Name:

-

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40 277/B
- Lot/batch No.: Vers. 1367/87 UO
- Expiration date of the lot/batch: August 1992
- Stabilty: Stable
- Stability of test article dilution: 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
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf, Switzerland
- Age at pretest: 8 weeks
- Weight at pretest: males: 166-189 g, females: 138 - 162 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ('Lignocel', Schill AG, 4132 Muttenz, Switzerland).
- Diet: Pelleted standard Kliba 343, Batch 82/87 rat maintenance diet ('Kliba', Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) available ad libitum
- Water: Community water from Itingen, available ad libitum
- Acclimation period: Seven days under laboratory conditions, after veterinary examination

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)

Remarks:

4% in distilled water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article dilutions were determined during pretest/acclimation. Intercurrent sampling for analyses was additionally performed during week 2 of test.

Duration of treatment / exposure:

up to 28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

30 males and 30 females;
Groups 0 mg/kg/day and 1000 mg/kg/day: 10 males; 10 females
Groups 50 mg/kg/day and 200 mg/kg/day: 5 males; 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

Based upon data received from acute studies and a 5-day oral toxicity (range-findings) study in rats (RCC Project 095220).

Examinations

Observations and examinations performed and frequency:

- Viability/mortality: Observations for mortality were recorded once daily.
- Clinical signs: Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.
- Food consumption: The food consumption was recorded once during the acclimatization period and weekly thereafter.
- Body weight: The body weight of each animal was recorded weekly during the acclimatization and treatment period.
- Ophthalmoscopic examinations: Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment. Ten minutes after the application of a mydriatic solution (Dispersa AG, Winterthur / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, Allschwil / Switzerland).
- Clinical laboratory investigations: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 7.00 and 9.30 a.m. to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retroorbital plexus. 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. Parameters being measured for hematology: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, Heinz-bodies, Methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, partial thromboplastin time. Parameters being measured for clinical biochemistry: glucose, urea, creatinine, bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total. Parameters being measured for urinalysis: volume (18 hour), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. The following organ weights were recorded on the scheduled date of necropsy: Adrenals, Aorta, Brain, Cecum, Colon, Cervix, 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, Gross lesions.

HISTOPATHOLOGY: Yes
- Slides of adrenals, heart, kidneys, liver, spleen, stomach and gross lesions (all animals) were collected at terminal sacrifice and from the animals of the control and high-dose groups examined by a pathologist. Upon detection of treatment-related morphologic changes in the organs of any high-dose animal, histologic evaluation of the same organs in all dose groups were performed.

Statistics:

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-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-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 for 2x2 tables was applied.

Results and discussion

Results of examinations

Details on results:

- Mortality: No death occurred.
- Clinical signs: Sedation was observed in two male rats of group 4 (1000 mg/kg bw) at day 8 of the treatment period. No other clinical signs were noted in the control or test article treated animals during treatment and recovery period.
- Food consumption: No significant differences in food consumption were observed during treatment and/or recovery period between the animals of the control and test article treated groups.
- Body weights: No significant differences in body weights were observed during treatment and/or recovery period between the animals of the control and test article treated groups.
- Ophthamlnoscopic examinations: No treatment-related findings were noted at termination of treatment and/or recovery period.
- Clinical laboratory investigations: The assessment of hematological, biochemical and urinalysis data indicated no changes of toxicolglcal significance at the end of the treatment, nor at the end of the treatment-free (recovery) period. However, a slightly higher incidence of proteinuria was noted for both sexes of animals in the 1000 mg/kg bw group after 4 weeks. This findings in the absence of morphological changes in the kidney, is considered to be a transient physiological proteinuria and not of toxicological relevance. Treatment-unrelated: All other differences in the results of the hematology, clinical biochemistry and urinalysis parameters were considered to be incidental and of normal biological variation. Based on other hematological data in the present study and on historical data, it can be concluded that there was no effect on Heinz body and methemoglobin formation after 4 weeks of treatment.
- Organ weights: After the 4 week treatment period, the female rats of group 4 (1000 mg/kg bw) showed statistically significant increased liver weight and ratios when the results were compared with those findings of groups 1 (control), 2 (50 mg/kg bw) and 3 (200 mg/kg bw). After 6 weeks the ratios from testes of male rats of group 4 were statistically significant decreased when compared with the controls. These differences were considered to be incidental and of no toxicological relevance.
- Macroscopic findings: Treatment-related yellowish discoloration of the gastro-intestinal mucosa was recorded in a number of rats of groups 3 and 4. Moreover, a few spontaneous gross lesions were encountered in both control and treated rats. These lesions were correlated with their histologic diagnoses.
- Microscopic findings: A treatment-related minimal fine granular brownish pigmentation of proximal convoluted tubules was recorded in the kidneys of four males and five females of group 4 at the end of the administration period. Spurious pigmentation was still evident in the kidneys of five females at the end of the recovery period. Enhanced epithelial desquamation of the gastrointestinal mucosa was recorded in a few rats of group 4 which had been examined for macroscopic abnormalities. Moreover, a few spontaneous microscopic lesions were recorded at a random incidence in both control and treated rats.

Effect levels

Key result

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: Effect level based on increased liver weight and enhanced epithelial desquamation of the gastro-intestinal mucosa.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL was determined to be 200 mg/kg/bw.

Executive summary:

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (0, 50, 200, and 1000 mg/kg bw) by repeated oral gavage, for a period of 28 days. The study was comprised of 4 groups, the groups comprised 5 animals per sex that were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. No treatment related effects were observed on mortality, food consumption, body weight, ophthalmoscopic examinations, and clinical laboratory investigations. Effects were limited to the high dose group (1000 mg/kg bw) and included sedation in two male rats at day 8, increased liver weight and ratios in female rats, minimal pigmentation of proximal renal tubules and enhanced epithelial desquamation of the gastro-intestinal mucosa. Therefore, the NOAEL was determined to be 200 mg/kg/bw.