Pentasodium 4-amino-6-[[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulphonatophenyl]azo]-3-[(2,5-disulphonatophenyl)azo]-5-hydroxynaphthalene-2,7-disulphonate

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

FAT 45168/A

IUPAC Name:

FAT 45168/A

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 ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4 4414 Fuellinsdorf/Switzerland
- Weight at study initiation: Males: 170.30 - 195.27 g; Females: 115.82 - 161.15 g
- Housing: Individually in Makrolon type-3 cages with autoclaved standard softwood bedding ('Lignocel', Schill AG, CH-4132 Muttenz).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: From 19-May-1994 to 25-May-1994 under laboratory conditions, after veterinary examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 %
- Air changes: 10 -15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light. 12 hours dark, music during the light period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled water

Details on oral exposure:

Dose levels
Group 1 (control): 0 mg/kg bw/day
Group 2: 50 mg/kg bw/day
Group 3: 200 mg/kg bw/day
Group 4: 1000 mg/kg bw/day

Test Article Preparation:
The test article 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 homogenizer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
Vehicle Bi-distilled water Frequency of preparation Daily prior to each application Chemical analysis of dose preparation
Concentration, homogeneity and stability of the test article/vehicle mixtures were determined during acclimatization. Further samples for analysis were taken during week 3 of the test and subsequently analyzed. The analyses were performed in the Analytical Laboratories of RCC Umweltchemie AG, according to a method which was supplied by the sponsor.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not available

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 and 1000 mg/kg per day
Basis:
no data

No. of animals per sex per dose:

Groups 1 and 4: 10 males; 10 females
groups 2 and 3: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based upon data from acute oral toxicity study (RCC Project 371777) and a 5-day dose-range finding study (RCC Project 371834) in which FAT 45'168 / A was administered orally by gavage to rats.

Positive control:

Not available

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS & MORTALITY:
Observations for mortality/viability and clinical signs of toxicity were recorded once daily.

BODY WEIGHT: The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a
Mettler PM 4800 balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION: Yes
Dates: at 4 weeks: 20-0UN-1994
at 6 weeks: 04-JUL-1994
A description of any abnormality was recorded. 10 - 90 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine BETA 200 Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil).

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

Sacrifice and pathology:

NECROPSY
after 4 weeks - 23-JUN-1994
after 6 weeks - 07-JUL-1994
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for
approximately 18 hours, but free access to water was provided. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. At the end of the treatment or recovery period the animals designated according to the necropsy schedule were anesthetized by
intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of 2.0 ml/kg body weight (equivalent to 320 mg sodium pentobarbitone/kg body weight), weighed and sacrificed by exsanguination. Samples of the following tissues and organs were collected from all animals at
necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution:
Adrenals; aorta; bone (sternum, femur); bone marrow (sternum, femur); brain; cecum; colon; duodenum; epididymides; esophagus; eyes with optic nerve and Harderian gland; femur including joint; heart; ileum; jejunum; kidneys; larynx; lacrimal gland, exorbital; liver; lung infused with formalin; lymph nodes,
mandibular, mesenteric; mammary gland area; nasal cavity; ovaries; pancreas; pituitary gland; prostate gland; rectum; salivary gland, (mandibular, sublingual); seminal vesicles; sciatic nerve; skeletal muscle; skin; spinal cord, (cervical, midthoracic, lumbar); spleen; stomach; testes; thymus; thyroid incl.
parathyroid gland; tongue; trachea; urinary bladder infused with formalin; uterus; vagina and gross lesions.
ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland. The organ to terminal body weight ratios as well as the organ to brain weight ratios determined:
The determination of the terminal body weight was performed immediately prior to necropsy using an on-line electronic recording system consisting of a Mettler PM 4000 balance connected to a computer system.
HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology were processed, embedded, cut at an approximate thickness of 4 micrometers, and stained with hematoxylin and eosin.
HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver, lungs, spleen, stomach and testes collected at terminal sacrifice from the animals of the control and high-dose groups were examined by a pathologist. The same applied to all gross lesions from all rats and to all animals which died spontaneously or have to terminated in extremis. All abnormalities were described and included in the report.

Other examinations:

Not available

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights, all ratios and clinical laboratory data :
When 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 for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to the ophthalmoscopy data.

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 specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

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:

CLINICAL SIGNS AND MORTALITY
No animal died during the course of study.
During the treatment period blue coloring of the feces was observed in animals of all treated groups.
There were no other treatment-related findings.

BODY WEIGHT AND WEIGHT GAIN
There were no effects on the mean body weight or body weight development in any dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Absolute and/or relative food consumption were increased in males and females of group 4 (1000 mg/kg bw/day) during the third and fourth week of treatment.
No test article related changes were observed in any other group.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmoscopic findings.

HAEMATOLOGY
The assessment of hematological data indicated the following effects in rats of groups 3 (200 mg/kg bw/day) and/or 4 (1000 mg/kg bw/day) at termination of the treatment:
- Slightly increased reticulocyte count (relative/absolute) in both sexes of group 4 with a tendency towards higher values in group 3, slightly increased HFR and slightly decreased LFR reticulocyte fluorescence ratio in males of group 4, and markedly increased methemoglobin (MET-HB) concentration in both sexes of group 4 with a slight increase observed in group 3. In addition, an increased incidence of polychromatophilia was noted in both sexes of group 4.
The findings were considered to be treatment-related and suggest methemoglobinemia with an increase in hemopoietic activity as indicated by the increase
in reticulocytes. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls.

CLINICAL CHEMISTRY
In the assessment of clinical biochemistry data the following effects were recorded in rats of groups 3 (200 mg/kg bw/day) and/or 4 at termination of the treatment:
- Slightly increased urea level in females (group 4), slightly increased creatinine level in both males and females (group 4), markedly increased uric acid and total bilirubin level in both males and females (group 4) and slightly increased in both males and females (group 3), slightly increased total cholesterol level in both males and females (group 4), slightly decreased triglyceride level in both males and females (group 4), slightly increased phosphorus level in females (group 4), marginally increased chloride level in females (group 4), slightly increased albumin and total protein level in both males and females (group 4), and slightly increased albumin to globulin ratio in males (group 4). In addition, a dark blue discoloration of the plasma was observed in all animals of group 4 and a light blue discoloration in all animals of group 3. These findings were considered to be test article related. At termination of the treatment-free recovery period most of these findings were found to be reversed and comparable to those of the controls, except for a slightly increased uric acid and total bilirubin level in both sexes and a slightly increased phosphorus level in females of group 4. Moreover, a light blue discoloration of the plasma was yet to be noted in all animals of this group.

URINALYSIS
Urinalysis data indicated no changes which could be considered of toxicological significance at termination of the treatment. The only alterations noted were a
deep brown to brown urine discoloration in both sexes of group 4 (1000 mg/kg bw/day) and a deep yellow urine pigmentation in three males and five females of group 3 (200 mg/kg bw/day). Also noted was a slight increase in the urine pH in females of group 4, slight increase in urine protein in both males and females of group 4 and marked increase in urine bilirubin (scores) in both males and females of group 4 with a slight increase in group 3. The discoloration was an expected effect of the test article, whereas the increase in urine bilirubin suggests a false positive reaction due to interference of the test article or metabolites thereof with the reagent test-strip reaction. This was also supported by the fact that "Ictotest" used to confirm positive bilirubin reaction was found to be negative in all cases nor was this latter observation supported by any underlying morphological findings which would suggest disturbed bilirubin metabolism, liver dysfunction or biliary obstruction. At termination of the treatment-free recovery period these findings were generally reversed and comparable to those of the controls, except for a slight increase in urine bilirubin and a yellow grey urine discoloration in both sexes of group 4.

ORGAN WEIGHTS
In comparison with the control group, no treatment-related effects on the organ weights or organ weight ratios were noted.

GROSS PATHOLOGY
At necropsy general and kidney discoloration (bluish) was observed in animals of group 3 and 4 (200, 1000 mg/kg bw/day). This was reversible after cessation of
treatment. At histopathological examination slightly increased extramedullar haematopoesis was observed in 2 males and 1 female of group 4 (1000 mg/kg bw/day) at the end of the treatment period and also in 1 male each of group 1 and 4 (0, 1000 mg/kg bw/day) after recovery.
No other treatment-related macroscopic or microscopic findings were noted.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The "no-observed-adverse-effect level" of FAT 45168/A is 50 mg/kg bw/day for male and female rats when administered orally by gavage for a period of 28 days.

Executive summary:

In this subacute toxicity study, FAT 45168/A was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. 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.

The only toxicologically significant observation during the in-life phase was an increase in food intake noted for both sexes of group 4 (1000 mg/kg bw/day) during the third and fourth week of treatment. At clinical laboratory investigations a number of slight to marked effects on biochemical, hematological and urinary parameters were noted for rats of group 4 (1000 mg/kg bw/day) and - in some cases - also of group 3 (200 mg/kg bw/day). In conclusion, these findings reflect methemoglobinemia with an increase in hemopoietic activity and with the corresponding histopathological findings of slightly increased splenic extramedullar hematopoiesis.

Based upon the results obtained in this study, the "no-observed-adverse-effect level" of FAT 45168/A is 50 mg/kg bw/day for male and female rats when administered orally by gavage for a period of 28 days.