2-{4-[4-[4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazin-2-ylamino]phenylazo]phenylazo}naphthalene-4,6,8-trisulfonate, trisodium salt

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:

27 April 2001 to 22 June 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals : Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-230-8

EC Name:

-

Cas Number:

321679-52-1

Molecular formula:

Hill formula: C31 H25 F N9 Na3 O12 S4 CAS formula: C31 H28 F N9 O12 S4 · 3 Na

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40'800/A
- Lot/batch No.: WP 2/01
- Purity: approx. 70%
- Stability: Stable under storage conditions
- Expiry date: 14 February 2007
- Storage: at room temperature (17-23°C) away from direct sunlight

Specific details on test material used for the study:

Identity: FAT 40800/A
Batch number: WP 2/01
Active ingredient: ca. 70 %
Aggregate state at room temperature: Solid / powder
Color: Dark orange
Stability of test item: Stable under storage conditions
Storage conditions: At room temperature at about 20 °C, away form direct sunlight
Expiration Date: February 14, 2007

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 6 weeks
- Weight at study initiation: Males: 130-154 g (mean 141 g), Females: 114 -133 g (mean 123 g)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet: Pelleted standard Provimi Kliba 3433 (batch no. 07/00 and 71/01) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

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:

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 (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC 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 (Group 1)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low dose (Group 2)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Middle dose (Group 3)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High dose (Group 4)

No. of animals per sex per dose:

The number of rats assigned to toxicity and recovery testing per group were:
- 5 per sex/dose for the toxicity test
- 5 per sex for 0 and 1000 mg/kg bw for the recovering test

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of a non-GLP 5-day dose range-finding study (RCC Study Number 806512) in which FAT 40800/A was administered by gavage to 2 rats per group and sex. Animals showed no reaction to treatment.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Observations for mortality/viability were recorded twice daily. The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).
DETAILED CLINICAL OBSERVATIONS:
- The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter
BODY WEIGHT:
- Body weights were recorded weekly during pretest, treatment and recovery and before necropsy.
FOOD CONSUMPTION:
- The food consumption was recorded once during the pretest period and weekly thereafter.
CLINICAL LABORATORY INVESTIGATIONS:
- Blood and urine sampling: after 4 and 6 weeks. Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a microhematocrit glass capillary tube. Urine was collected during the 18-hour fasting period into a specimen vial.
HAEMATOLOGY: Parameters being measured: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin,
Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Reticulocyte fluorescence ratios, Nucleated erythrocytes (normoblasts), Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, Thromboplastin time (=prothrombin time), Activated partial thromboplastin time.
CLINICAL CHEMISTRY: Parameters being measured: Glucose, Urea, Creatinine, Total bilirubin, Total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium
Phosphorus, Sodium, Potassium, Chloride, Albumin, Total Protein, Globulin, Albumin/Globulin ratio
URINALYSIS: Parameters being measured: Volume (18-hour), Specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Nitrite, Urobilinogen, Urine sediment, Red blood cells, White blood cells, Crystals (Triple phosphate).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Grip strength: Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AGF 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.
- Locomotor activity: Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were randomized and monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

Sacrifice and pathology:

GROSS PATHOLOGY:
- Sacrifice after 4 and 6 weeks. All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Ovaries, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
HISTOPATHOLOGY:
- Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution: Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (cerebrum, cerebellum, pons), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions.
- Slides of all organs and tissues listed were collected (except: Aorta, Bone, Esophagus, Eyes with optic nerve, Harderian gland, Larynx, Lacrimal gland, Mammary gland area, Nasal cavity, Pancreas, Pituitary gland, Salivary glands, Skeletal muscle, Skin and Tongue) at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (stomachs) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as clinical laboratory data: The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex or Student's t-test, as appropriate. 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.
Fisher's exact-test was applied to the macroscopic findings. Armitage/Cochran Trend Test was used for non-neoplastic lesions, if appropriate.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs noted in males and females treated with 1000 mg/kg/day included discolored feces and orange bedding (beginning on treatment day 2). These findings were considered to be passive test item-related changes commonly seen following oral administration of dyestuffs, rather than indications of systemic toxicity. In one male treated with 1000 mg/kg/day, localized alopecia and scabbed wounds (beginning on treatment day 2) were noted. These findings were considered to result from internecine conflict and were isolated occurrences unrelated to the treatment with the test item. No test item-related clinical signs of toxicological relevance were noted at any dose level during daily observations. In males treated with 1000 mg/kg/day, miosis was noted in one male during week 2 and mydriasis was noted in one male during week 3. These findings were, in view of their isolated occurrence, considered to be incidental. No clinical signs were noted in females at any dose level during weekly observations (weeks 1-3).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After four weeks' treatment, the red blood cell count of the females treated with 1000 mg/kg/day was significantly lower (p <0.01) than that of the control females. In the absence of similar changes in related parameters, this difference was considered to be incidental. The relative reticulocyte count of the males treated with the 1000 mg/kg/day was significantly higher (p <0.05) than that of the controls. In the absence of a dose-response relationship in the males treated with 50 mg/kg/day and 200 mg/kg/day, this finding was considered to be incidental.
The methemoglobin level of the test item-treated males was significantly higher (p <0.05 or p <0.01) than that of the controls, but this difference was considered to be due to a low control value. The methemoglobin level of the test item-treated females was unaffected. In the absence of similar changes in related parameters, or a clear dose-response relationship, all other differences to the control values were considered to be unrelated to the test item treatment.
After two weeks' recovery, only the slightly elevated methemoglobin levels noted in males previously treated with 1000 mg/kg/day remained. All of the above-mentioned differences were reversible. The absolute leukocyte count of the males previously treated with 1000 mg/kg/day was slightly elevated (p<0.05) when compared with the control; this difference was primarily due to an elevated lymphocyte count (p <0.05) and, in the absence of similar changes in the females, was considered to be incidental. In females previously treated with 1000 mg/kg/day, a marginal (but statistically significant, p <0.05) reduction in the mean cell hemoglobin concentration remained within the 95 % tolerance limits of the historical control data and was considered to be of no toxicological significance. Similarly, a marginal reduction in the relative count of segmented neutrophils of females treated with 1000 mg/kg/day was considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total globulin was lower in the males and females treated with 1000 mg/kg/day when compared with the respective controls. Although statistically significant only in males (p <0.01 ), both values were outside the 95 % tolerance limits of the historical control data and therefore considered to be test item-related. Albumin levels were unaffected, but the albumin/globulin ratios were minimally elevated in the males and females treated with 1000 mg/kg/day. These differences attained statistical significance (p <0.05) in males. After four weeks' treatment, males treated with 1000 mg/kg/day had significantly increased (p <0.01) activity of lactate dehydrogenase when compared with the control males. This difference, however, was not supported by a dose-response relationship (males treated with 50 mg/kg/day had clearly higher activity than males treated with 200 mg/kg/day), nor did it exceed the 95 % tolerance limits of the historical control data, and therefore was considered to be incidental. The activity of lactate dehydrogenase was significantly lower (p <0.05 or p <0.01) in females of all dose levels, but this difference was considered to be caused by a high control value. The activity of alanine aminotransferase was significantly reduced (p<0.05) in females treated with 1000 mg/kg/day, but remained within the lower range of the 95 % tolerance limits. All other differences in the clinical biochemistry parameters after four weeks' treatment either remained within the range of the 9 5% tolerance limits of the historical control data, were isolated changes unsupported by concomitant changes in related parameters, or were without a clear dose-response relationship and therefore considered to be incidental. After the two-week recovery period, all differences to the control values noted in the clinical biochemistry parameters of rats previously treated with 1000 mg/kg/day remained within the range of the 95 % tolerance limits and therefore considered to be incidental.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

After four weeks' treatment, ketone was present in the urine of females treated with 200 mg/kg/day and in both sexes treated with 1000 mg/kg/day. This finding was considered to be test item-related. All other differences in the urinalysis parameters were not dose related and therefore considered to be incidental. After the two-week recovery period, significantly increased (p <0.05) urinary output, significantly reduced specific gravity and osmolality (both p <0.05) was noted in males previously treated with 1000 mg/kg/day. These differences were within the range of the 95 % tolerance limits of the historical control data and therefore considered to be incidental.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In one male treated with 1000 mg/kg/day, localized alopecia and scabbed wounds were noted during the functional observational battery (week 4). All other animals were without clinical signs during week 4.

Grip Strength
The forelimb grip strength of the males treated with 50 mg/kg/day was significantly higher (p<0.01) and the hindlimb grip strength of the females treated with 50 mg/kg/day was significantly lower (p<0.05) than that of the respective control values. All other values compare favorably with those of the controls. In the absence of similar changes at 200 mg/kg/day or 1000 mg/kg/day, these differences were considered to be incidental.

Locomotor Activity
From 0-15 minutes, the mean locomotor activity of the males treated with 1000 mg/kg/day was significantly lower (p<0.01) than that of the control males. The total mean locomotor activity (0-60 minutes) was slightly lower in the males treated with 1000 mg/kg/day when compared with the controls. This change was, in the absence of similar changes in the females, considered to be test item-unrelated. The mean locomotor activity of the test item-treated females compared favorable with that of the control females.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

After 4 Weeks
No test item-related differences were noted at any dose level tested. The significantly increased spleen-to-body weight ratio (p <0.01) noted in the males treated with 200 mg/kg/day was not dose related and therefore considered to be unrelated to treatment.

After 6 Weeks
The absolute and relative organ weights of the rats previously treated with the test item compared favorably with those of the controls.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Changes considered to be associated with the administration the test item were present in the stomach of both sexes given 1000 mg/kg/day. No test item-related histopathological findings were present in animals given 50 or 200 mg/kg/day. All of the other microscopic findings encountered were considered to have arisen spontaneously.
Stomach: Minimal or slight degenerative changes in the mucus neck cells of the glandular mucosa, characterised by the presence of brightly eosinophilic cytoplasmic inclusions in the mucous neck cells and increased basophilia and some depletion of mucous in the foveolar cells, was present in five males and five females given 1000 mg/kg/day. This finding was accompanied in four males and a female by minimal focal submucosal inflammatory cell infiltration. Neither of these findings occurred in animals from any of the other groups on study. Minimal or slight focal spongiosis of the squamous epithelium at the limiting ridge was present in four males and four females given 1000 mg/kg/day and in a male and a female control. In addition, minimal focal erosion of the glandular stomach and minimal focal epithelial hyperplasia at the limiting ridge were observed in a female and a male, respectively, given 1000 mg/kg/day.
These findings were considered to indicate that the test item had caused local irritation of the gastric mucosa. The changes seen in the stomach at the end of the dosing period were also present in animals allowed a treatment free period of 14 days. However, given that the most likely explanation for the occurrence of these findings was local irritation by the test item it is considered that with sufficient time all the changes would resolve.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

urinalysis

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) of FAT 40800/A was determined to be 200 mg/kg bw/day.

Executive summary:

In a GLP-compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (50, 200, and 1000 mg/kg bw) 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. For the post-exposure recovery period two satellite groups exposed to 0 and 1000 mg/kg bw for 28 days consisting of five male and five female rats each were monitored for an additional 14 days after the end of the 28 days treatment period. No treatment related effects were observed on mortality, clinical signs, grip strength, locomotor activity, food consumption, body weight, organ weight and macroscopic findings. Clinical laboratory investigations showed that total globulin was lower after 28 days treatment in the males and females treated with 1000 mg/kg/day when compared with the respective controls, and that ketone was present after four weeks treatment in the urine of females treated with 200 mg/kg bw and in both sexes treated with 1000 mg/kg bw. Microscopically, treatment-related findings were diagnosed in the stomach in rats treated with 1000 mg/kg bw where minimal or slight focal spongiosis in the forestomach, minimal or slight degenerative changes in the mucus neck cells of the glandular stomach, minimal focal submucosal inflammatory cell infiltration in the glandular stomach; and minimal focal erosion in a female and minimal focal epithelial hyperplasia at the limiting ridge in a male were observed. After 14 days recovery, there was little evidence of reversibility. Nonetheless, given that the likeliest etiology of the lesion was local irritation it is probable that with sufficient time the findings would resolve. Based on the study results, the no observed adverse effect level (NOAEL) of FAT 40800/A was determined to be 200 mg/kg bw/day.