disodium 7-{[4-chloro-6-(dodecylamino)-1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[{4-[(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

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:

from January 10 to February 28, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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)

Version / remarks:

(1981)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: revised Japanese chemical substance law (1987)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Red GS 3848

IUPAC Name:

Red GS 3848

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animalsSource: BRL Biological Research Laboratories Ltd., SwitzerlandAge at study initiation: 6 weeksWeight at study initiation: 147.6 - 160.6 g (males); 132.0 - 145.1 g (females)Housing: individually in cagesDiet: ad libitumWater: ad libitumAcclimation period:6 days, under laboratory conditions, after veterinary examination. Only animals without any visual signs of illness were used.Environmental conditionsTemperature: 20 - 22 °CHumidity: 50 - 58 %Air changes: 10 - 15 per hourPhotoperiod: 12 hours artificial fluorescent light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Preparation of dosing solutionsThe substance was weighed into a glass beaker on a tared Mettler PM 480 balance and the vehicle was added. The mixture (w/v) was prepared using a homogeniser. Homogeneity of the test article in the vehicle was maintained during the treatment using a magnetic stirrer.Frequency of preparationDaily prior to each application.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of test article/vehicle mixtures were determined during acclimatisation. Further samples for analysis were taken during week 3 of the test and subsequently analyzed.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily, 7 days per week for a total of 28 days.

Doses / concentrations

Remarks:

Doses / Concentrations:0, 50, 200, 1000 mg/kg dayBasis:actual ingested

No. of animals per sex per dose:

Group 1 (control): 10/sexGroup 2 (50 mg/kg): 5/sexGroup 3 (200 mg/kg): 5/sexGroup 4 (1000 mg/kg): 10/sex

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based upon data from acute oral toxicity study dose level selection and a 5-day dose range-finding study in which the substance was administered orally by gavage to rats.

Positive control:

No data.

Examinations

Observations and examinations performed and frequency:

Mortality and clinical signs: yesTime schedule: once dailyBody weight: yesBody weight of each animal was recorded once during the acclimatisation period and weeekly thereafter. Additionally, terminal body weights were recorded at necropsy.Food consumption: yesFood consumption was recorded once during the acclimatisation period and weekly thereafter.Ophtalmoscopic examination: yesTime for examinations: at 4 weeks (all animals); at 6 weeks ( on all recovery animals of group 1 and 4)Dose groups that were examined: 0, 50, 200, 1000 mg/kgHematology: yesTime for collection of blood: after 4 weeks and after 6 weeks, between 7 and 8.30. Anaesthetic used for blood collection: slight ether anesthesiaAnimals fasted: yes, for 18 hours with access to water ad libitumHow many animals: all animalsParameters checked: RBC, HB, HCT, MCV, MCH, MCHC, platelet count, reticulocyte count, reticulocyte fluorescence ratios, NEN, HEINZ-BOD, MET-HB, WBC, differential WBC, red cell morphology, PT, APTT.Clinical biochemistry: yesTime for collection of blood: after 4 weeks and after 6 weeks, between 7 and 8.30. Anaesthetic used for blood collection: slight ether anesthesiaAnimals fasted: yes, for 18 hours with access to water ad libitumHow many animals: all animalsParameters checked: glucose, urea, creatinine, uric acid, total bilirubin, total cholesterol, triglycerides, phospholipids, ASAT/GOT, ALAT/GOT, ALAT/GPT, LDH, CK, ALP, G-GT, calcium, phosphorous, sodium, potassium, chloride, albumin, total protein, globulin, albumin/globulin ratio.Urinalysis: yesTime for collection of urine: after 4 weeks and after 6 weeks, during the 18-hours fasting periodParameters checked: volume (18 hours), specific gravity, osmolality, colour, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment.Neurobehavioural examination: no data

Sacrifice and pathology:

Gross pathologyAll animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected at necropsy: adrenals aorta, bone, bone marrow, brain, cecum, colon, duodenum, epididymes, esophagus, eyes with optic nerve and Harderian gland, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland, exorbital, liver, lung, lymph node, mandibular, mesenteric, mammary gland area, nasal cavity, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary gland, seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina and gross lesions.HistopathologySlides of adrenals, heart, kidneys, liver, lungs, spleen, stomach and testes colIected at terminal sacrifices from the animals of the control and high-dose groups were examined.

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

Description (incidence and severity):

Statistically significant increase of ASAT and ALAT activity in female of 1000 mg/kg group, but reversible. No toxicological significance is cosidered to be associated with these changes.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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:

MortalityNo test article related deaths were noted in this study. One female of group 4 (1000 mg/kg), died on day of scheduled necropsy after blood sampling. This death was considered to be incidental.Clinical signsNo test article related clinical signs of reaction to treatment were noted in all groups.Food consumption/ Relative food consumptionMale and female animals in all dose groups (50, 200 and 1000 mg/kg) had similar food consumption and relative food consumption to those recorded in the respective control animals. No test article related effects were evident.Body weights In alI groups, mean body weights and body weight gain were similar. Up to and including the highest dose level of 1000 mg/kg, the treatment with the test article did not affect the body weight development of animals.Ophthalmoscopic examinationsFew abnormal findings noted, in terms of persistent pupillary membrane in two control animals and in one group 2 (50 mg/kg) female and corneal opacity in one group 4 (1000 mg/kg) female, gave no indications of test article related effects.Hematology/clinical biochemistry/urinalysisAssessment of hematological, clinical biochemistry and urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. The only change of note, was a marginal but statistically significant increase of ASAT and ALAT activity in female rats of group 4 (1000 mg/kg) at termination of the treatment. These findings were found to be reversible at termination of the treatment-free recovery period. However, statistical significance at the 5 % level was still noted for ASAT. No toxicological significance is associated with these changes. In addition, the findings were well within limits of the historical control data and there were no morphological findings which would suggest a treatment related effect. All other statistical differences in results of clinical Iaboratory data were considered to be incidental or unrelated to the treatment and of normal biological variation for rats of this strain and age. Organ weights, organ to body weight and organ to brain weight ratiosDifferences in organ weights noted between control animals and animals of the dose groups (50, 200 and 1000 mg/kg) were not considered to be caused by treatment with test article. The few statistically significant differences noted were considered to be incidental.Macroscopic and microscopic findingsIn group 4 (1000 mg/kg), discoloration caused by test article in various segments of the intestinal tract was noted. In addition, one male of this dose group had red-brown discoloration of the lungs. At histopathological examination, this finding was diagnosed as multifocal macrophage accumulations which contained dyestuff particles. This finding was also noted in one other group 4 (1000 mg/kg) male and was presumed to be due to accidental inhalation of the test article.The remainder of macroscopic and microscopic findings were considered to be spontaneous in nature. Neither the types nor the incidences distinguished control animals from treated animals.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all tested parameters

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a 28-day study in rats via oral gavage, the substance does not show adverse effects up to the highest tested dose of 1000 mg/kg bw/day.

Executive summary:

Method

Subacute toxicity study. Daily administration of the substance by gavage to Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 days. The number of animals in each group was 10, 5, 5 and 10 per sex, respectively. After termination of the treatment period half of the animals of the control group and of the high dose group were observed for a further 14 -day treatment-free recovery period.

Result

Up to and including the highest dose level of 1000 mg/kg, there was no test article related deaths or clinical signs of reaction to-treatment and no effects on the food consumption or body weight development of the animals. At ophthalmoscopic examinations, no test article related abnormal findings were noted. The recording of the organ weights gave no indications of test article related effects.

The assessment of hematological , clinical biochemical and urinalysis data indicated a single change of note: at 1000 mg/kg, there was a marginal but statistically significant increase of the aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activity in female rats at termination of the treatment. These findings were found to be reversible at termination of the treatment-free recovery period. No toxicological significance is considered to be associated with these findings.

At macroscopical and microscopical examination, there were no morphological alterations which gave evidence of toxic effects.

At 1000 mg/kg, discoloration caused by the test article in various segments of the intestinal tract was noted. In addition, one male had red-brown discoloration of the lungs. At histopathologic examination, this finding was diagnosed as multifocal macrophage accumulations which contained dyestuff particles. The same finding was also noted in one other male of the same group and was presumed to be due to accidental inhalation of the test article.

Based upon these results, the no-observed-adverse-effect level was considered to be 1000 mg/kg body weight for male and female rats when administered orally by gavage.