Reaction mass of Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-7-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate and Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-6-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate

Repeated dose toxicity: oral

Some information in this page has been claimed confidential.

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 45'168 / A
- Purity: ca. 80%
- Lot/batch No.: 316904.36
- Expiration date of the lot/batch: June 1998
- Stability of test article dilution: FAT 45'168/A was found to be stable in bi-distilled water at room temperature for at least two hours.
- Storage condition of test material: at room temperature in the dark.

Specific details on test material used for the study:

None

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

As per guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, 4414 Fuellinsdorf/Switzerland
- Age at delivery: 6 weeks
- Body Weight and Range at acclimatization/pretest: 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 (e.g. ad libitum): Pelleted standard Kliba no. 343, Batch nos. 73/94 and 76/94, rat maintenance diet ('Kliba', Klingentalmuehle AG, CH-4303 Kaiseraugst) ad libitum.
- Water (e.g. ad libitum): community tap-water from Itingen was available ad libitum.
- Acclimation period: from May 19, 1994 to May 25, 1994 under laboratory conditions, after veterinary examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12, music during light period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Bi-distilled water

Details on oral exposure:

DOSE VOLUME: 10 mL/kg body weight per treatment day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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.

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:
50, 200, 1000 mg/kg per treatment day
Basis:
actual ingested

No. of animals per sex per dose:

30 males, 30 females;
groups 0 mg/kg bw and 1000 mg/kg bw: 10 males; 10 females
groups 50 mg/kg bw and 200 mg/kg bw: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

In this subacute toxicity study, FAT 45'168/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. The dose was 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. 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 treatmentfree recovery period.

Examinations

Observations and examinations performed and frequency:

MORTALITY / VIABILITY AND CLINICAL SIGNS
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
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
Dates: at 4 weeks: 20-JUN-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, CLINICAL CHEMISTRY, URINALYSIS :
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted in metabolic cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected between the hours of 07.00 and 08.30 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-hours fasting period into a specimen vial.
Blood and urine sampling: after 4 weeks 23-June-1994, after 6 weeks 07-July-1994

The following parameters were determined:
HAEMATOLOGY:
Erythrocyte count (RBC), Hemoglobin (HB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELETS), Reticulocyte count (RETIC.), Reticulocyte fluorescence ratios (HFR = high, MFR = middle, LFR = low), Nucleated erythrocytes (normoblasts) (NEN), Heinz bodies (HEINZ-BOD.), Methemoglobin (MET-HB), Total leukocyte count (WBC), Differential leukocyte count (Diff. WBC Count), Red cell morphology, Thromboplastin time (= Prothrombin time) (PT), Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY:
Glucose, Urea, Creatinine, Uric acid, Bilirubin total (BILI T.), Cholesterol total (CHOLEST. T.), Triglycerides (TRIGL.), Phospholipids (PHOS.LIPID), Aspartate aminotransferase (ASAT/GOT), Alanine aminotransferase (ALAT/GPT), Lactate dehydrogenase (LDH), Creatine kinase (CK), Alkaline phosphatase (ALP), Gamma-glutamyltransferase (G-GT), Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein total, Globulin, Albumin/Globulin ratio (A/G RATIO).

URINALYSIS:
Volume (18-hour), Specific gravity (SPEC. GRAV.), Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, , Urobilinogen (UROBILI.), (SED. MICRO.).

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.

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

Description (incidence and severity):

There were no test article related clinical signs except for blue coloring of the feces in all treated groups. These findings were reversible within 1 to 4 days after cessation of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on the mean body weight or body weight development in any dose group. In males of group 4 (1000 mg/kg) mean body weight was slightly but consistently lower when compared with the control group (0 mg/kg). Since the difference attained statistical significance only at week 2 of treatment and body weight gain was similar in all groups, these findings are considered to be incidental and to lie within the normal range of biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (absolute and relative) was in the same range for animals of the control group 1 (0 mg/kg) and treated groups 2 and 3 (50, 200 mg/kg). In group 4 (1000 mg/kg) a statistically significant lower food intake (absolute) was noted for males during the first week of treatment whereas statistically significant higher food intake was noted for males during week 3 (relative) and week 4 (absolute and relative), and for females during week 3 and 4 (absolute and relative). These differences in food consumption disappeared during the first week of the recovery period and are considered to be related to the test article.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmoscopic findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the assessment of the hematological data the following statistically significant effects were recorded in rats of groups 3 (200 mg/kg) and/or 4 (1000 mg/kg) at termination of the treatment:
- Slightly increased reticulocyte count (relative/absolute) by 26 to 29 % on average in both sexes of group 4. There was also an apparent tendency towards higher values for group 3, however, this group did not attain statistical significance.
- Slightly increased HFR reticulocyte fluorescence ratio by 48% and slightly decreased LFR fluorescence ratio by 9 % on average in males of group 4.
- Markedly increased methemoglobin (MET-HB) concentration by 383 to 373 % on average in both sexes of group 4 and slightly increased by 59 to 81 % in both sexes of group 3.
- Slightly higher incidence of polychromatophilia in both sexes of group 4, however, the increase did not attain statistical significance. The changes noted were considered to be treatment-related and reflect 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. All other differences in the results of the hematology parameters were considered to be incidental and unrelated to the treatment, and of normal biological variation for rats of this strain and age.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

CLINICAL BIOCHEMISTRY
For clinical biochemistry data the following effects were recorded in rats of groups 3 (200 mg/kg) and/or 4 (1000 mg/kg) at termination of the treatment:
- Slightly increased urea level by 24 % on average in females of group 4.
- Slightly increased creatinine level by 15 to 12 % on average in both sexes of group 4.
- Markedly increased uric acid level by 861 to 763 % on average in both sexes of group 4 and slightly increased by 108 to 109 % in both sexes of group 3.
- Markedly increased total bilirubin level by 603 to 611 % on average in both sexes of group 4 and slightly increased by 72 to 87 % in both sexes of group 3.
- Slightly increased total cholesterol level by 15 to 35 % on average in both sexes of group 4.
- Slightly decreased triglyceride level by 33 to 73 % on average in both sexes of group 4.
- Slightly increased phosphorus level by 15 % on average in females of group 4.
- Marginally increased chloride level by 1 % on average in females of group 4.
- Slightly increased albumin level by 13 to 10 % on average in both sexes of group 4.
- Slightly increased total protein level by 9 % on average in both sexes of group 4.
- Slightly increased albumin to globulin ratio by 8 % on average in males of group 4.
Also noted was a dark blue discoloration of the plasma in all animals of group 4 and a light blue discoloration in all animals of group 3. At termination of the treatment-free recovery period most of these findings were found to be reversed and comparable to those of the controls. However, the following changes were yet to be noted in the animals of group 4:
- Slightly increased uric acid level by 37 % on average in males and 89 % in females, whereby the increase in males did not reach statistical significance.
- Slightly increased total bilirubin level by 68 to 78 % on average in both sexes.
- Slightly increased phosphorus level by 25 % on average in females.
In addition, all animals of this group indicated a light blue discoloration of the plasma.
The findings were considered to be test article related. However, considering the discoloration of the plasma, the groups affected and the magnitude of change in two of the parameters measured (uric acid and total bilirubin), these findings suggest interference by the test article. As for total bilirubin part of the increase may have been related to a greater erythrocyte turnover (hemoglobin liberation) as a result of the methemoglobinemia, whereas the other changes suggest metabolic adaptations. All other differences in the results of the clinical biochemistry parameters were considered to be incidental and unrelated to the treatment, and of normal biological variation for rats of this strain and age.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis data indicated no changes of toxicological significance at termination of the treatment, however, the following alterations were noted when compared to the controls:
- Deep brown urine discoloration in ten males and eight females and brown discoloration in two females of group 4 (1000 mg/kg) and deep yellow urine pigmentation in three males and five females of group 3 (200 mg/kg).
- Slight increase in urine pH in females of group 4.
- Slight increase in urine protein (scores) in both sexes of group 4.
- Marked increase in urine bilirubin (scores) in both sexes of group 4 and slight increase in both sexes of group 3.
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. The abnormally deep yellow, brown, deep brown and yellow-grey pigmentation of the urine was an expected effect of the test article, whereas the higher bilirubin scores apparently reflect false positive reactions 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 reactions was found to be negative in all cases. In addition, the observation was not supported by any underlying morphological findings which would suggest disturbed bilirubin metabolism, liver dysfunction or biliary obstruction. It may be noted that normally, urine does not contain any bilirubin. Free or unconjugated bilirubin which results from the breakdown of hemoglobin is not water soluble, is bound to plasma proteins and is not excreted in the urine, whereby conjugated bilirubin (bilirubin glucuronide) which is water soluble, is excreted in the urine. All other differences in the results of the urinalysis parameters were considered to be incidental and unrelated to the treatment, and of normal biological variation for rats of this strain and age.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In comparison with the control group, no treatment-related effects on the organ weights or organ weight ratios were noted for all dose groups. Statistically significant differences consisted of lower testes weight in group 2 (50 mg/kg), higher spleen to brain weight ratio in males of group 3 (200 mg/kg) and higher adrenals weight in females of group 4 (1000 mg/kg). These findings are considered to be incidental and of normal biological variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy bluish discoloration of the kidneys and also of general tissues in animals of group 3 and 4 (200, 1000 mg/kg) was noted which was clearly attributable to the treatment with test article. Since there were no histopathological counterparts to these findings, they are considered to be the result of colored test article remnants and/or metabolites in the excretory organs (kidneys) and other tissues. All other alterations observed are considered to be incidental and of normal biological variation.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The assessment of microscopic findings is based on the histopathological examination of adrenals, heart, kidneys, liver, lungs, stomach and testes from animals of groups 1 and 4 (0, 1000 mg/kg), and of spleen and gross lesions from animals of all groups. Treatment-related findings consisted of slightly increased extramedullar haematopoiesis observed in group 4 (1000 mg/kg) from 2 males and 1 female. In the recovery groups 1 male was affected each in group 1 (0 mg/kg) and group 4 (1000 mg/kg). No other treatment-related findings were noted. A number of minor degenerative and inflammatory lesions were observed which fell within the normal range for rats of this strain and age.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

urinalysis

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The NOAEL of the test substance 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 a GLP-compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats of both sexes were treated with the read across substance at dose levels of 0, 50, 200 and 1000 mg/kg bw/day for a period of 28 days by oral gavage. Dose groups 0 and 1000 mg/kg bw/day containing 10 males and 10 females; and dose group 50 and 200 mg/kg bw/day containing 5 males and 5 females. 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. However, confirmatory histopathology, i.e. increased splenic extramedullary haematopoiesis, was confined to the high dose group (1000 mg/kg bw/day) and was only minimal to slight in degree in the affected animals. At termination of the treatment-free recovery period most of these hematological, biochemical and urinary 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; 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. During the treatment period blue coloring of the feces was observed in animals of all treated groups. At termination of treatment, 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. At necropsy general and kidney discoloration (bluish) was observed in animals of group 3 and 4 (200, 1000 mg/kg), this was reversible after cessation of treatment. Based upon the results obtained in this study, the "no-observed-adverse-effect level" of the test substance is 50 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days.