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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experiment start date - 01 April 1999; Experiment completion date - 20 May 1999; Study completion date - 07 July 1999.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-240-8
EC Name:
-
Cas Number:
212652-59-0
Molecular formula:
Hill formula: C25 H22 F N8 Na3 O13 S4 CAS formula: C25 H25 F N8 O13 S4 · 3 Na
IUPAC Name:
trisodium 3-amino-4-[2-(4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}-2-sulfonatophenyl)diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate
Specific details on test material used for the study:
Identity: FAT 40574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf / Switzerland
Age at delivery: 6 weeks
Body weight range at acclimatization: Males: 135 - 151 grams (mean 142 grams), Females: 111 - 134 grams (mean 121 grams).
Identification: Cage card and individual ear tattoo
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, with target ranges for room temperature 22 ± 3°C and relative humidity 40-70% (values above 70% during cleaning process possible).
Room environment was monitored continuously with hourly recordings. The room was illuminated by fluorescent light on a 12 hour light/dark cycle.
Accommodation: Groups of three in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)
Diet: Pelleted standard Kliba 3433, batch no. 35/98, rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum (except for the overnight fasting period prior to intubation).
Water: Community tap water from Itingen, available ad libitum.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
water
Remarks:
Bi-distilled
Details on oral exposure:
Method of administration: Gavage
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 on the first day of treatment. 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:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group - Group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low dose group - Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Mid dose group - Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose group - Group 4
No. of animals per sex per dose:
Male: 10 animals at 0 mg/kg bw/day
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 200 mg/kg bw/day
Male: 10 animals at 1000 mg/kg bw/day
Female: 10 animals at 0 mg/kg bw/day
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 200 mg/kg bw/day
Female: 10 animals at 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3 (target: ca 1 and 3 hours after administration); as well as once daily on days 4-28 (target: ca. 1 hour after administration), and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether 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 micro-hematocrit glass capillary tube.
Urine was collected during the 18-hour fasting period into a specimen vial.
after 4 weeks: 06 May 1999
after 6 weeks: 20 May 1999 (post recovery)
The assays were performed at RCC Ltd (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weights were recorded weekly during pretest, treatment and recovery and before each necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether 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 micro-hematocrit glass capillary tube.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin, concentration, Platelet count, Reticulocyte count, Reticulocyte fluorescence, Nucleated erythrocytes (normoblasts), Heinz bodies, Methaemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, Thromboplastin time (=prothrombin time), Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Early morning
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked: Plasma color, Glucose, Urea, Creatinine, Uric Acid, Bilirubin, total Cholesterol, total Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein, total Globulin, Albumin/Globulin ratio

NEUROBEHAVIOURAL EXAMINATION: Yes
During week 4, relevant parameters (presented in Appendix I) from a modified Irwin screen test were evaluated in all animals.
The test was performed approximately 1 hour after application. NB. The results of the Functional Observational Battery are presented in the summary and individual tables of the Detailed Clinical Observations (Weekly) under week 4. This method of data presentation permits a clear evaluation and assessment of weekly clinical signs observed during the study.
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 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.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
after 4 weeks: 06 May 1999
after 6 weeks: 20 May 1999 (post recovery)

All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist.
All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution. Following is list of organs evaluated for gross pathology
Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (telencephalon, 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

HISTOPATHOLOGY: Yes
Slides of all organs and tissues listed in boldface type (see Necropsy, above) which were collected at scheduled sacrifices from the animals of control and high-dose groups were examined by a pathologist. All gross lesions from the animals of the low and middle dose group as well as heart and colon (males only), stomach and cecum from males and female rats of these groups were processed as described under Histotechnique and examined by light microscopy.
Statistics:
The following Statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all 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.
• 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 macroscopical findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test article-related signs were noted in any animal during weeks 1-3.
Mortality:
no mortality observed
Description (incidence):
All animals survived the scheduled study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of the animals of test article-treated groups were generally similar to those of the control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption and relative food consumption of the test article-treated animals compared favorably with that of the controls.
Haematological findings:
no effects observed
Description (incidence and severity):
The hematology parameters were considered to be unaffected by the test article.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Plasma glucose levels were decreased in all animals treated with 1000 mg/kg/day, and cholesterol (males and females) triglyceride (males) and phospholipid levels (males) were elevated when compared with the control values. The differences attained statistical significance (Dunnett test, p <0.05 or 0.01) but were considered to be metabolic adaptation to the administration of a xenobiotic. All other differences to the control values were considered to be fortuitous.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis parameters were considered to be unaffected by the test article.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute and relative organ weights of the test article-treated animals compared favorably with those of the controls (treatment and recovery).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Discoloration of various organs was observed in the high dose group in males and females. This is considered rather to be an effect of treatment with a dye stuff rather than a toxic effect. Other isolated findings did not differ treated rats from the controls and were within the historical range of findings commonly seen in animals of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A number of microscopic findings were considered to distinguish rats treated with the test article at 1000 mg/kg from those of the control group at the end of the treatment period:
• Heart: increased progressive myocardiopathy in males;
• Stomach: increased hyperkeratosis of non-glandular stomach, increased eosinophilic inflammatory infiltration;
• Cecum erosion in one male rat, inflammation, associated with congestion and edema, in one male and in one female rat;
• Colon: inflammation in one male rat.
The findings observed in the heart and in the stomach persisted until the end of the recovery period of 14 days, in addition to glandular stomach erosion in one female treated with 1000 mg/kg/day.
All other microscopic findings noted at the end of treatment or recovery were considered to be incidental findings commonly occurring in rats of this strain and age.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
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:
The no-observed-adverse-effect-level (NOAEL) and as the no-observed-effect level (NOEL) was established to be 200 mg/kg bw.
Executive summary:

In this subacute toxicity study, FAT 40574/B was administered daily by oral gavage to SPF bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. This study was conducted according to OECD test guideline 407 in a GLP-certified laboratory. A control group was treated concurrently with the vehicle. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, stomach and cecum (males and females) as well as the heart and colon (males) were examined to establish a no-effect level. The oral administration of FAT 40574/B to Wistar rats at doses of 50, 200 and 1000 mg/kg/day for 28 days did not affect food consumption or body weight development, nor were any clinical signs of toxicity evident. No test article-related deaths occurred. Haematology and urinalysis parameters were unaffected. Organ weights were unchanged by treatment. There were no findings at necropsy which were considered to be of toxicological significance. In animals treated with 1000 mg/kg, plasma glucose levels were decreased, whereas cholesterol (in both sexes), phospholipid (in males) and triglyceride levels (in males) were elevated when compared with the control values. These findings are considered to be metabolic adaptation to the administration of a xenobiotic. Test article-related findings of toxicological significance were restricted to microscopical changes at 1000 mg/kg/day in the heart (males only) and stomach (males and females). These test article-related findings noted at the end of the treatment were not reversible during the 14-day recovery period. Based on the results of this study, 200 mg/kg body weight/day of FAT 40'574/B was established as the no-observed-adverse-effect-level (NOAEL) and as the no-observed-effect level (NOEL).