Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

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:

06 January 1987 to 02 February 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

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))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

EC Number:

402-170-5

EC Name:

Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

Cas Number:

106359-91-5

Molecular formula:

C26H26FN9O13S4.3Na

IUPAC Name:

trisodium 7-{2-[2-(carbamoylamino)-4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}phenyl]diazen-1-yl}naphthalene-1,3,6-trisulfonate

Details on test material:

- Name of test material (as cited in study report): FAT 40'224/C
- Description: powder
- Lot/batch No.: Op. 1/86; Vers. 10/86
- Expiration date of the lot/batch: Oktober 1991
- Stability of test article dilution: stable for at least 2 hours
- Storage condition of test material: room temperature in the dark; protected from humidity

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Op. 1/36, Vers. 10/86
- Expiration date of the lot/batch: October, 1991

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: 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 study initiation: 7-8 weeks
- Weight at study initiation: males: 154 - 177 g, females: 156 - 178 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ('Lignocel', Schill AG, 4132 Muttenz, Switzerland).
- Diet: Pelleted standard Kliba no. 343, Batch 57/86 and 62/86 rat maintenance diet ('Kliba', Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) ad libitum.
- Water: Community tap water from Itingen was 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

Details on route of administration:

Oral by gavage, once daily.
The control animals received the vehicle without the test article.
Rationale: Accidental oral ingestion is a possible route of human exposure.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

4 % in distilled water

Details on oral exposure:

Dose volume: 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, stability and homogeneity of the test article/vehicle mixtures was determined before test initiation and at week 2 of test. Analyses were performed in the RCC Analytical Laboratory, according to a method supplied by the sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

FAT 40224/C was administered to SPF-bred Wistar rats by repeated oral gavage, for a period of 28 days. The study was comprised of four groups, each containing five male and five female rats. The test article/vehicle mixture was administered on a mg/kg bw basis by oral gavage (50, 200, 1000 mg/kg bw). The dose was based upon data received from a 5-day oral toxicity (range-finding) gavage study. The animals of the control group were treated similarly with the vehicle alone.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Observations for mortality were recorded daily

DETAILED CLINICAL OBSERVATIONS: Yes
Signs of toxicity were assessed daily. A description of any abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
The body weight of each animal was recorded weekly during the acclimation and treatment period using an on-line electronic recording system consisting of a Mettler PK 4800 balance.

FOOD CONSUMPTION: Yes
The food consumption was recorded once during the acclimation period and weekly thereafter using an online electronic recording system consisting of a Mettler PK 4800 balance.

OPHTHALMOSCOPIC EXAMINATION: Yes
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).

HAEMATOLOGY/ CLINICAL BIOCHEMISTRY: Yes
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 biologic variation caused by circadian rhythm. Blood samples were drawn from the retro-orbital plexus.
Blood sampling: after 4 weeks

- Parameters being measured: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes normoblasts, total leukocyte count, differential leukocyte count, red cell morphology, coagulation, thromboplastin time, partial thromboplastin time, glucose, urea, creatinine, bilirubin total, cholesterol total, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total.

URINALYSIS: Yes
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: after 4 weeks

- Parameters being measured: volume (18 hour), specific gravity, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organ weights were taken from all animals necropsied at termination of treatment: Adrenal glands, kidneys, liver, testes.

Complete necropsies were performed on all rats. Representative tissue specimens from the following organs were taken and fixed in 4% phosphate buffered neutral formaldehyde solution: Adrenal glands, kidneys, liver, and any gross lesions.

HISTOPATHOLOGY: Yes
All tissue samples as listed above from all rats were trimmed, embedded in paraffin wax, sectioned at a nominal thickness of 4 microns and stained with hematoxylin and eosin. Sections were also prepared from all tissues with abnormal macroscopic findings.

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-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. 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

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs and mortality which could be related to test article application were observed during the study period.
One female, 1000 mg/kg bw group died spontaneously during anesthesia for terminal blood collection.

BODY WEIGHT/ BODY WEIGHT GAIN
No statistically significant differences in body weight gains were observed between the animals of the test article-treated and control groups.

FOOD CONSUMPTION
No statistically significant differences in food consumption were observed between the animals of the test article-teated and control groups.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings were noted.

HAEMATOLOGY/ CLINICAL CHEMISTRY/ URINALYSIS
The assessment of hematological, biochemical and urinalysis data indicated no changes of toxicologicai significance between treated rats and controls, at the end of the treatment.
All differences in the results of the hematology, clinical biochemistry and urinalysis parameters were considered to be incidental and of normal biological variation.

ORGAN WEIGHTS AND ORGAN WEIGHT RATIOS
No statistically significant differences in absolute and relative organ weights and ratios were observed between the animals of the test article-treated and control groups.

GROSS PATHOLOGY
No treatment related macroscopic findings were recorded

HISTOPATHOLOGY:
No treatment related microscopic findings were recorded. The various spontaneous microscopic findings recorded are within the normal range observed in this age and strain of rat. They may be attributed to subclinical illness, spontaneous congenital abnormalities or physiological status.

Effect levels

Key result

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: no treatment related adverse effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The no-toxic effect level of FAT 40224/C in 28 day repeated dose toxicity study in rats is 1000 mg/kg bw.

Executive summary:

The subacute 28-day study with FAT 40224/C was carried out in Wistar rats according to OECD guideline 407 and EU method B.7 as per GLP compliance. The test substance administered daily by gavage to SPF-bred Wistar rats. The study was comprised of four groups, each containing 5 male and 5 female rats. The dose levels were administered are 0, 50, 200 and 1000 mg/kg body weight. No deaths which could be related to test article application occurred during the course of the study. Animal No. 38 (female, group 4) died spontaneously during anesthesia for terminal blood collection. No clinical signs which could be related to test article application were observed during the study period. No statistically significant differences in food consumption and body weight gain were observed between the animals of the test article-treated and control groups. No treatment-related findings were noted. The assessment of hematological, biochemical and urinalysis data indicated no changes of toxicological significance between treated rats and controls, at the end of the treatment. No statistically significant differences in organ weights and organ to body weight ratios were observed between the animals of the control and test article treated groups. All pathology findings recorded were of a spontaneous nature common to rats of this age and strain. There was no evidence of abnormal histopathological findings resulting from treatment with FAT 40224 / C. Based on the results obtained in this study, the "no-toxic effect- level" of FAT 40224/C is greater than 1000 mg/kg body weight for male and females rats when administered orally by gavage.