tetrasodium 2-(4-fluoro-6-(methyl-(2-(sulfatoethylsulfonyl)ethyl)amino)-1,3,5-triazin-2-ylamino)-5-hydroxy-6-(4-methyl-2-sulfonatophenylazo)naphthalene-1,7-disulfonate

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:

July 08, 1999 to Aug. 19, 1999

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

432-550-6

EC Name:

-

Cas Number:

243858-01-7

Molecular formula:

Hill formula: C25H22FN7Na4O16S5 CAS formula: C25H26FN7O16S5.4Na

IUPAC Name:

tetrasodium 2-({4-fluoro-6-[methyl({2-[2-(sulfonatooxy)ethanesulfonyl]ethyl})amino]-1,3,5-triazin-2-yl}amino)-5-hydroxy-6-[2-(4-methyl-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Reaktiv Orange F97-0318

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARLAN WINKELMANN, Gartenstr. 27, 33178 Borchen, SPF breeding colony
- Age at study initiation: approximately 6 weeks
- Housing: in makrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniffl R/M-H (V 1534), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ±20%
- Photoperiod: 12 h light/12 h dark

IN-LIFE DATES: From: July 08, 1999 To: Aug. 19, 1999

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Remarks:

first and last week of treatment

Details on analytical verification of doses or concentrations:

HPLC of samples from all dose groups

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Acute oral toxicity testing of the test substance at a dose of 2000 mg/kg in the rat (only this dose was tested) showed that the LD50 is above 2000 mg/kg bw in both male and female animals. The dose of 2,000 mg/kg was tolerated by all the animals without signs of intoxication.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL EXAMINATION: Yes
- Time schedule: Once before the first treatment and thereafter once a week

FOOD CONSUMPTION: Yes
- Time schedule: 2 times per week

WATER CONSUMPTION: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the study and then twice weekly

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period,
- Anaesthetic used for blood collection: Yes ((intraperitoneal injection of 67 + 6. 7 mg/kg bw Ketamine-Hydrochloride + Xylazine).
- Animals fasted: No
- Hematology investigations included erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin {MCH), mean corpuscular hemoglobin concentration (MCHC), leukocyte count, thrombocyte count, differential leukocyte count and red cell morphology,reticulocyte count, Heinz bodies, coagulation time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Animals fasted: No
- How many animals: 5/sex/dose
- Clinical chemistry parameters evaluated consisted of sodium, potassium, inorganic phosphorus, uric acid, bilirubin total, bilirubin direct, creatinine, glucose, urea, calcium, chloride, aspartate aminotransferase (ASA T/GOT), alanine aminotransferase (ALA T/GPT), alkaline phosphatase CAP),
gamma-alutamvltranspeptidase (GGT), cholesterol, triglycerides, total protein and albumin.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 27 for treatment groups and Day 41 for recovery groups
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Urinalysis consisted of following investigations: appearance, color, volume, specific weight, pH, hemoglobin, protein, glucose, bilirubin, urobilinogen, ketone bodies and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the termination of the study.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other: sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive), presence and absence of pupillary constriction, measurement of motor activity, and forelimb and hindlimb grip strength.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. skin, orifices, eyes, feeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes. Brain, adrenal glands, ovaries, heart, stomach, uterus, lungs, jejunum, lymph nodes (iliac), trachea, colon, lymph nodes (mandibular), thymus, urinary bladder, thyroid with parathyroid glands, liver, testes, bone marrow (sternum), spleen, epididymides, N. ischiadicus , kidneys,prostate gland, spinal cord (cervical).

Other examinations:

ORGAN WEIGHTS: The following organs were weighed and the organ to body weight ratios calculated: heart, liver, kidneys, adrenal glands, spleen, testes, epididymides, thymus and brain.

Statistics:

Treatment group values were compared statistically with the control group values at the level of significance p = 0.05

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy of the final and recovery value some animals of the intermediate and nearly all animals of the high dose group showed red or brown-red discolored kidneys. Additionally, all males of the terminal and three males of the recovery high-dose group showed light red discolored testes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed intratubular deposition of yellow-red pigment in the kidneys of nine and three animals of the high-dose terminal sacrifice and recovery groups, respectively. This finding was a result of deposition of test material in the tubular cells, particularly in the high dose terminal sacrifice group. This substance deposition did not cause morphological changes in the kidneys, consequently, it was considered to be of no toxicological relevance. In the stomach dose dependent submucosal mixed cellular infiltrations were found in all animals of the terminal sacrifice high-dose group, and in a lower degree in eight intermediate-dose rats. This was considered to be the result of a topical but reversible acute irritation caused by the high salt load administered with the test substance.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No clinical signs were observed. No mortality occured throughout the study.

BODY WEIGHT AND WEIGHT GAIN: Body weight development remained unaffected by the administration of the test substance in all groups.

FOOD CONSUMPTION: Food consumption of all animals remained unaffected by the administration of the test substance throughout the study.

WATER CONSUMPTION: There was no alteration for water consumption throughout the study.

HAEMATOLOGY: A significant increase of the platelets in males of the high dose group was observed. This finding was not considered to be treatment related as it was not accompanied by a statistically significant decrease in coagulation time, occurred in one sex only, and could not be observed in the recovery group. All other hematological parameters remained unaffected.

CLINICAL CHEMISTRY: Clinical chemistry analysis revealed that the serum was discolored orange in all animals of both sexes from the high-dose group. This discoloration was believed to impair the measurement of total bilirubin and total protein and consequently, caused a false significant deviation from the control group. The clinical chemistry also revealed statistically significant increase in serum calcium and cholesterol in female animals of the high dose group. Total protein and albumin was also indicated to be increased in females of the intermediate and/or low dose groups, respectively. Additionally, a decrease in potassium, triglycerides, and glucose was noted in intermediate and high-dose females. Low dose females also had low glucose level. The analyses at the end of the recovery period indicated a statistically significant increase of sodium, potassium, phosphorus, total protein, and albumin in females and sodium, chloride, creatinine, and ASA T in males of the high-dose group. A decrease was noted in the glucose value in males. The increase in serum calcium, cholesterol, and albumin in after 28 d of treatment was considered to be not related to the administration of the test substance. These apparent statistical significances were caused by the lower values of the control animals compared to the historical control data. The apparently increased parameters were found to be inside the physiological ranges and correspond with the historical control data. The decrease in potassium, triglycerides, and glucose in treated animals were also considered incidental findings as all values were in the physiological range of the historical control data.
Consequently, the changes in serum parameter were considered to be of no biological significance and not to be substance related. This was also confirmed taking into consideration that there were no corresponding findings in the recovery group.

URINALYSIS: The urine examination did not show any compound-related effect, sediments were inconspicuous.

NEUROBEHAVIOUR: Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, and forelimb and hindlimb grip strength were not influenced by the administration of the test compound in all groups. As a result of the lower standard deviation of this group, the statistical evaluation revealed an increased motor activity in females of the high dose group. However, this finding was considered not to be treatment-related, as it occurred in one sex only, on average activity of the females was lower in the high than in the intermediate dose group, and the historic control values of males and females were about in the same range.

ORGAN WEIGHTS: At the end of the 28 d treatment period, the statistical evaluation revealed a significant increase of the absolute liver weight of intermediate and high dose females. In relative organ weights a statistically significant increase in spleen weight or kidney weights was noted in males or females, respectively. In males of the high-dose recovery group an increase of the relative liver weight was found. The apparent changes in mean organ weight were considered to be incidental and mainly caused by the lower terminal mean body weight of concerned groups.

GROSS PATHOLOGY: At necropsy of the final and recovery value some animals of the intermediate and nearly all animals of the high dose group showed red or brown-red discolored kidneys. Additionally, all males of the terminal and three males of the recovery high-dose group showed light red discolored testes.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathological examinations revealed intratubular deposition of yellow-red pigment in the kidneys of nine and three animals of the high-dose terminal sacrifice and recovery groups, respectively. This finding was a result of deposition of test material in the tubular cells, particularly in the high dose terminal sacrifice group. This substance deposition did not cause morphological changes in the kidneys, consequently, it was considered to be of no toxicological relevance. In the stomach dose dependent submucosal mixed cellular infiltrations were found in all animals of the terminal sacrifice high-dose group, and in a lower degree in eight intermediate-dose rats. This was considered to be the result of a topical but reversible acute irritation caused by the high salt concentration administered with the test substance. Further, sporadic findings were observed in various organs of different animals, which correlated to the gross pathology findings (pigment deposits in the lymph nodes); type and incidence of these changes were not relevant for the assessment of the toxicity of the test substance.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the test conditions, No Observed Adverse Effect Level (NOAEL) for systemic effects, No Observed Adverse Effect Level (NOAEL) for local effects and No Observed Effect Level (NOEL) were determined to be 1000 mg/kg bw, 62.5 mg/kg bw and 62.5 mg/kg bw, respectively.

Executive summary:

A 28 d oral (gavage) repeated dose toxicity study was conducted in Sprague Dawley rats according to OECD guideline 407 and EU method B.7 in compliance with GLP.

Four groups of animals (5/sex/dose) received the test substance at the dose of 0.0, 62.5, 250.0 and 1,000.0 mg/kg bw/day by oral gavage for a period of 28 d. 14 d recovery groups (control and high dose) were also included in the study. No clinical signs and no mortality occurred throughout the duration of study. No test substance related effects were seen for body weight, food and water consumption, organ weights, hematology, clinical chemistry and urinanalysis investigations. Histopathology rexamination revealed reversible dose dependent, slight acute topical irritations in the submucosa of the stomach in intermdeiate and high dose animals. No systemic effects were observed.

Under the test conditions, No Observed Adverse Effect Level (NOAEL) for systemic effects, No Observed Adverse Effect Level (NOAEL) for local effects and No Observed Effect Level (NOEL) were determined to be 1000 mg/kg bw, 62.5 mg/kg bw and 62.5 mg/kg bw, respectively.