Benzenesulfonic acid, 2-[2-[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]diazenyl]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:2)

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:

21 April 1993 to 19 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OCED test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Details on test material:

See below

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test species: Wistar rat
Strain: Hoe: WISKf(SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Age at start of study: approx. 6 weeks
Animal maintenance: in fully air-conditioned rooms in Makrolon cages (Type 4) on soft wood granulate in groups of 5 animals
Room temperature: 22 ± 3⁰C
Relative humidity: 50 ± 20 %
Lighting time: 12 hours daily
Acclimatization: approx. 5 days
Food: Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe), ad libitum except for the period in which the animals were kept in diuresis cages
Water: tap water in plastic bottles, ad libitum except for the period in which the animals were kept in diuresis cages
Animal identification: fur-marking with KMn04, numbered ear tags and cage numbering
Randomisation: Allocation of animals to cages: Randomisation tables 93.177 and 93.178 (detailed in the report).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionised

Details on oral exposure:

Route of application: orally gavage
Vehicle: deionised water

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

Treatment group Dose
(mg/kg bw./d) No. of animals Animal Nos.
Male Female Male Female
1 0 5 5 1-5 21-25
2 62.5 5 5 6-10 26-30
3 250 5 5 11-15 31-35
4 1000 5 5 16-20 36-40

Control animals:

yes, concurrent no treatment

Details on study design:

Rationale for dose-selection:
Testing of Reaktiv-Gelb F-66 923 FW for acute oral toxicity in the Wistar rat yielded a median lethal dose above 2000 mg/kg bodyweight. After application of 2000 mg/kg bodyweight the animals showed squatting posture, stilted gait as well as mucous and yellow discoloured feces. No signs of intoxication were observed one day after application.
Based on these results Reaktiv-Gelb F-66 923 FW was tested in the present study at the dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day.

Rationale for the route of exposure: The oral route is considered to be a potential exposure route in man.

Rationale for species selection: The Wistar rat has proved to be a suitable species for subacute oral toxicity testing with many different substances.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

Behaviour and State of health
The behaviour and general health condition of the animals were observed twice daily, and at weekends and on public holidays once daily. The animals were examined weekly for neurological disturbances, opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

Body weight
The body weights of all animals were determined at the start of the study and then twice weekly throughout the study.

Food and water consumption
Food consumption was determined continuously (2 times per week). The values on the printouts refer to the intervals between one measurement and the next. They are converted to the food consumption per 100 g bodyweight over a 24 hour period.
Water consumption was determined once weekly over a period of 16 hours and is given in the results as water consumption / animal / 16 h (from 3.15 p.m. to 7.15 a.m.)

Hematological investigations
At the termination of the study, hematological examinations were performed on all animals without previous withdrawal of food. Blood samples were taken from the retroorbilal venous plexus in narcosis (intraperitoneal injection of 50 -100 mg Ketamin / kg bodyweight). In order to prevent systematic errors, blood sampling was conducted in a randomised order.

The following hematological parameters were determined:

Erythrocyte count, Hemoglobin, Hematocrit, Mean cellular volume (MCV), Mean cellular haemoglobin (MCH), Mean cellular haemoglobin concentration (MCHC), Leucocyte count, Thrombocyte count, Differential leucocyte count and red cell morphology count*, Heinz bodies*, Coagulation time.

*These parameters were scored only in animals of the control and high dose group.

Clinical Chemistry
After blood sampling for hematological testing, the animals were killed by cutting of the vena cava cranial is in deep narccsis and exsanguinated. In order to prevent systematic errors, exsanguination was conducted in a randomised order.

The following serum values were determined:

Sodium,Potassium, Inorganic phosphorus, Uric acid, Bilirubin total, Creatinine, Serum-glucose, Urea nitrogen, Chloride, Aspartate aminotransferase (ASAT/G0T), Alanine aminotransferase (ALAT/GPT), Alkaline phosphatase (AP), Gamma-glutamyltransferase (GGT), Cholesterol, Triglycerides, Total protein, Albumin.

Urine analysis
Urine analysis was performed on all animals a few days before termination of the study, for this purpose, the urine was collected by using metabolism cages (overnight night from day 26 to 27).

The following parameters were checked:

Appearance, Colour, pH-Value, Hemoglobin, Protein, Glucose, Ketone bodies, Bilirubin, UrobiIinogen, Specific Weight, Sediment*, Volume.

*These parameters were scored only in the control and high dose group

Sacrifice and pathology:

Necropsy and macroscopic, examination
After exsanguination, all animals were necropsied in accordance with the Dissection Schedule I of the Department for Pathology and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs. All abnormal findings were recorded.

Organ weights
The following organs were weighed and the organ to body weight ratio calculated:
Heart, Liver, Kidneys, Ovaries, Spleen, Adrenals, Testes, Epididymides.

Histopatholoqy
The following tissues or organs (or pieces of them) were preserved in a suitable fixative in accordance with the Dissection Schedule of the Department for Pathology and processed for histopathological investigations:
Heart, Liver, Kidneys, Adrenals, Spleen, Testes, Stomach, Jejunum, Colon, Uterus, Ovaries, Epididymides

Statistics:

The following parameters were compared statistically with the control group values at the level of significance p = 0.05:
Body weights at the designated measurement times
Hematological data
Clinical chemistry parameters
Urine analysis (Volume, pH-value and specific weight) Absolute organ weights and organ to body weight ratios

Evaluation was performed by Pharma Research and Development Informatics with the aid of a program package for the evaluation of toxicological studies, according to the respective standard operating procedures. The calculation methods used are referred to are detailed in their entirety within the report..

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional 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

Histopathological findings: neoplastic:

not examined

Details on results:

Behaviour, state of health and mortality
Behaviour and general health condition of the animals remained normal throughout the study.
Compound-related neurological disturbances, opacity of the refracting media of the eyes, impairment of dental growth or changes of the oral mucosa were not observed .
No deaths occurred throughout the study.

Body weight gain
Body weight development was not impaired by the administration of the test compound and was comparable in all groups.
Body weights were increased to a statistically significant degree in males of all treatment groups (low dose: day 6 until day 13; intermediate and high dose: day 1 until day 16).
Females of the high dose group showed statistically significant decreases in bodyweights before the start of the study.

Food and water consumption
Absolute and relative food consumption remained normal throughout the study.
Likewise, the administration of the test compound did not alter absolute or relative water consumption.

Hematological investigations
Statistical evaluation revealed increases in coagulation times in males of the intermediate and high dose group. There was no typical dose-dependency. Furthermore, the changes were only small and occurred in one sex only. Therefore, a compound-related effect is not directly evident. However, no adverse effects were observed in these animals.
Furthermore, hemoglobin levels were increased to a statistically significant degree in females of the high dose group. These values were within the normal range of the rat strain used [126-163 g/l] and low hemoglobin concentrations were measured in females of the control group. Therefore, a compound-related effect is unlikely.
Heinz bodies were not detected. Evaluation of differential white cell count revealed no abnormalities.

Clinical chemistry
Statistical evaluation revealed decreases in sodium and bilirubin values as well as increases in triglyceride concentrations in males of the high dose group. All values were within the normal range of the rat strain used [sodium; 135-151 mmoi/l; bilirubin: 1.3-6.1 umol/l; triglycerides: 0.52-2.67 mmol/l]. Furthermore, the changes occurred in one sex only. Therefore, a compound-related effect is not directly evident. However, no adverse effects were noted in these animals.
Females of the low dose group showed statistically significant increases in protein and albumin levels. As there was no dose-dependency, a compound-related effect can be ruled out.

Urine analysis
Examination of the urinary status revealed no compound-related effects, the sediments were inconspicuous.

Organ weights
Liver and kidney weights were increased in males of the intermediate and high dose group. There was no typical dose-dependency. Furthermore, these changes occurred in one sex only and there was no evidence for an impairment of liver or kidney function, neither by clinical chemistry nor by histopathology. Therefore, a compound-related effect is not evident.
Heart weights were increased to a statistically significant degree in males of the intermediate dose group. As there was no dose-dependency, a compound-related effect is unlikely.

Macroscopic and microscopic findings
No compound-related effects were observed at necropsy.
Histopathological examinations revealed no compound-related effects.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion Reaktiv-Gelb F-66 923 FW caused no detectable adverse effects in male and female Wistar rats when administered 28 times during 29 days. With regard to the present study the 'no observed adverse effect level' is 1000 mg/kg bodyweight per day.

Executive summary:

The present 28-day toxicity study was conducted in order to characterise the toxicological profile of Reaktiv-Gelb F-66 923 FW after repeated oral exposure. Additionally, the results of this study can be used as a dose-range-finding for subchronic and chronic feeding studies.  

The present study was conducted in compliance with OECD Guidelines for Testing of Chemicals, 407 "Repeated Dose Oral Toxicity -Rodent: 28-day or14-day Study" and in compliance with the Principles of Good Laboratory Practice (GLP).

 

Groups of 5 male and 5 female Wistar rats received Reaktiv-Gelb F-66 923 FW by oral gavage at dose levels of 0, 52.5, 250 or 1000 mg/kg body weight per day for 28 days and were necropsied at day 29.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly.

Hematological examinations and clinical chemistry were carried out at the termination of the study. Urine analysis was also performed at the end of the study.

During necropsy the animals were examined for microscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, urine data (volume, pH value, specific weight), absolute and relative organ weights were analysed with the aid of a statistical program to show differences compared with the controls .

 

Behaviour, state of health, body weight development, food and water consumption were not affected by the administration of the test compound in males and females from all groups.

Urine analysis, hematological examinations and clinical chemistry showed no compound-related effects.

Organ weights were not affected by the administration of the test compound. No compound related macroscopically visible changes were found at necropsy. Likewise, histopathological examinations revealed no substance-related changes.

 

In conclusion Reaktiv-Gelb F-66 923 FW caused no detectable adverse effects in male and female Wistar rats when administered 28 times during 29 days. With regard to the present study the 'no observed adverse effect level' is1000 mg/kg bodyweight per day.