REAKTIVE GELB F 97494

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:

From November 22, 2001 to January 03, 2002

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: OPPTS 870.3050

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-090-0

EC Name:

-

Cas Number:

477795-15-6

Molecular formula:

C26H24FN8Na3O14S4

IUPAC Name:

Trisodium 2-{2-[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazen-1-yl}-4-{[4-fluoro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reaktivgelb F-97494

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Standard animal for this kind of study

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Harlan Winkelmann GmbH, Gartenstrasse 27, D-33178 Borchen
- Age at study initiation: approximately 6 weeks
- Housing:In transparent macrolon® cages (type IV) on soft wood granulate in an air-conditioned rooms, 5 animals per cage, separated according to sex
- Diet: Standard diet for rats (ssniff® R/M-H (V 1534)), exept for the period in which the animals were kept in diuresis cages
- Water: Tap water in plastic bottles ad libitum, exept fot the period in which the animals were kept in diuresis cages
- Acclimation period: at least 5 d
- Animal identification: Tatto number on the tail and cage numbering
- Randomization procedure: Computer generated algorithm (archived with raw data)

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C (except short lasting deviations due to disturbances of air condition)
- Humidity: 50±20% (except short lasting deviations due to disturbances of air condition)
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: November 22, 2001 to January 03, 2002

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Administration volume: 5 ml/kg bw

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

-PREPARATION OF DOSING SOLUTIONS: test substance was dissolved in deionized water. After each measurement of the body weight, the calculation of the application volume was repeated.
- Concentration in Deionized water (vehicle) : 1.0, 4.0 and 16.0% w/v
- Application volume: 5 mL/kg bw
- Frequency of administrations: once daily

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparations were tested immediately after preparation and after two weeks. The results were within the range of 80 to 120% of the nominal concentration.

Duration of treatment / exposure:

28 d

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Five/sex: 50 and 200 mg/kg bw/day
Ten/sex: 0 and 800 mg/kg bw/day (Five/sex in recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

-Dose selection rationale: Acute oral toxicity testing of test substance at a dose of 2000 mg/kg in the rat (limit test) showed that the median lethal dose (LD50) is above 2000 mg/kg bw in both male and female animals. No lethality occurred after application of 2000 mg/kg bw. No symptons occurred in male animals. Female animals showed stilted gait and yellow-discolored urine 1 h up to 8 h after administration. Development of body weight was not impaired. The animals killed at the end of the observation period showed no macroscopically visible changes. Based on these results, dose levels of 0, 50, 200 and 800 mg/kg bw/day, were selected for the present study.

-Duration of recovery period: 14 d

-Test groups: At the beginning of the acclimatization period, the test animals were randomized and assigned to the respective groups (i.e., 0, 50, 200 and 800 mg/kg bw/day).

Examinations

Observations and examinations performed and frequency:

Mortality
Survival control of the animals was examined twice daily (on weekends and public holidays once daily).

Clincal observations
The behavior and general health condition of the animals were observed once daily.

Neurological examinations
Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Each animal was assessed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, salivation, nasal discharge, piloerection, pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, tremor, and any other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination were evaluated.

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

Food consumption
Food consumption was determined before the start of the study and then twice weekly throughout the study.

Clinical pathology
Hematological investigations
At the termination of the study and after the recovery period, hematological examinations were performed on all animals without previous withdrawal of food. Blood samples were taken from the retrobulbar venous plexus in narcosis (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine-Hydrochloride + Xylazine). In order to prevent systematic errors, blood sampling was conducted in a randomized order. Hematology parameters evaluated consisted of the following:
1) Red cell counts parameters (i.e., erythrocyte counts (RBC), hematocrit (packed cell volume), hemoglobin, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte counts and heinz body counts*)
* This paramenter was only evaluated in the animals from the control and high dose group.
2) White Cell Counts parameters (i.e., differential leukocyte counts, leukocyte counts (WBC))
3) Coagulation parameters (i.e., thrombocyte counts (platelets) and coagulation time (clotting time))

Clinical Chemistry
After blood sampling for hematological testing, the animals were killed by section of the vena cava cranialis in deep narcosis and exsanguinated. In order to prevent systematic errors, exsanguination was conducted in a randomized order.

Clinical chemistry parameters evaluated consisted of γ-glutamyltranspeptidase, alanine aminotransferase (ALAT), albumin, albumin / globulin ratio (calculated), alkaline phosphatase, aspartate aminotransferase (ASAT), bilirubin direct, bilirubin total, calcium, chloride (Cl-), cholesterol, creatinine, globulin (calculated), glucose, inorganic phosphorous, potassium (K+), sodium (Na+), total protein, triglycerides, urea, and uric acid

Urinalysis
Urine analysis was performed in all animals a few days before termination of the study. For this purpose, using metabolism cages (overnight from 26 d to 27 d) collected the urine. Food was withdrawn during this period.

Urinalysis consisted of semiquantitative parameters (i.e., appearance, bilirubin, blood, color, glucose, ketone bodies, microscopic examination (sediment)*, ph, protein, urobilinogen, volume, specific weight)
* This parameter was only evaluated in the animals from the control and high dose group.

Sacrifice and pathology:

Anatomic pathology

Necropsy and macroscopic examination
After exsanguination, all animals were necropsied 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.

Endotracheal fixation of the lungs: The lungs, including part of the trachea, were removed. The lungs were then fixed endotracheally with a formalin solution using a needle inserted into the trachea. The instillation pressure was between 20 and 30 cm water column. Following completion of the endotracheal fixation the lungs were fixed, together with the other organs, in formalin solution.

Organ weights
The organs were weighted and the organ to body weight ratios calculated for adrenals, brain, epidymides, heart, kidneys, liver, spleen, testes, and thymus

Macroscopic and microscopic observations
The tissues or organs (or pieces of them) were preserved in a suitable fixative and processed for histopathological investigations. The tissues or organs examined were adrenals, bone marrow / sternum, brain with medulla oblongata, epididymides, heart, small intestine to jejunum, large intestine 2 colon, kidneys, liver, lungs, lymph nodes 1 mandibular, lymph nodes to iliacal, nerve sciatic nerve, ovaries with oviducts, prostate, seminal vesicle, spinal cord 1 cervical, spleen, stomach, testes, thymus, thyroid gland with parathyroid, trachea, urinary bladder, uterus and all other gross lesions. Samples of organs mentioned above were embedded by conventional histological technique in Paraplast. All organs were stained with hematoxylin-eosin. In the absence of any specific target organs, histopathological examination was almost confined to the control and high dose animals, with the exception of the stomach, which was also examined for low and mid dose group animals.

Statistics:

All parameters: The assumption of a monotonic dose-response relationship for all parameters justifies the restriction of the significance level to 5 percent (per parameter and sex), using the method of: Hothorn L, Lehmacher W.: A Simple Testing Procedure "Control versus k Treatments" for One-sided Ordered Alternatives, with Application in Toxicology, Biom. J. 33, 179-189, Akademie Verlag

Bodyweights: The changes of parameter values compared to the treatment-free baseline values are analyzed. The baseline values themselves are not statistically evaluated, since by definition no treatment effect can be present at this stage. Method used: t-Test: Hartung J., Elpert B., Klosener K. H., Lehr- und Handbuch der angewandten Statistik (1989), R. Oldenbourg Verlag, Munchen

Clinical pathology data: In dog studies, the changes of parameter values compared to the treatment-free preliminary values are analyzed. The preliminary values themselves are not statistically evaluated, since by definition no treatment effect can be present at this stage. In rat studies, the absolute values are analyzed. Wilcoxon's Test: Hollander m., Wolfe, D. A:, Nonparametric statistical methods; Wiley Series in Probability and Mathematical Statistics (1973), John Wiley & Sons Inc., New York, with the exact distribution after Steitberg B, Rohmel J.: Exakte Verteilung fur Rang- und Randomisierungstests imallgemeinen Stichprobenproblem, EDV in Medizin und Biologie 18, 12-19 (1987), Verlag Eugen Ulmer GmbH & Co., Stuttgart; Gustav Fisher Verlag KG, Stuttgart

Organ weights (absolute): t-Test (Hartung J., Elpert B., Klosener K.H.,Lehr- und Handbuch der angewandten Statistik (1989), R. Oldenbourg Verlag, Munchen

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight was statistically significantly decreased in high dose males between study 5 and 29 d (ca. 10%, treatment period) and during recovery, when compared to the control. Mean overall body weight gain was reduced, accordingly.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

slightly but statistically significant increase in the urinary specific weight was observed in high dose males. However, the increase was very slight and within the range of biological variation and hence considered to be of no toxicological relevance.

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:

effects observed, treatment-related

Description (incidence and severity):

Distinct mixed-cellular infiltrations in the stomach was observed in the high dose group with an average grading of 3.2 (male)/3.5 (female), which was clearly reversible throughout the post treatment period.

Details on results:

Behavior, state of health and mortality:
One high dose female animal was killed for animal welfare reasons on 6 d of the study. A substance related cause of death was unlikely in the absence of any gross pathology findings and any histopathological findings in the lungs.

Clinical observation:
There were no clinical findings observed during the observation period in any dose group animals. As an exception, the high dose female animal, which had to be killed for animal welfare reasons, exhibited respiratory sounds from 2 d onwards with subsequentsigns of poor general condition, including hypoactivity, stilted gait and squatting posture. In the absence of any clinical symptoms in all remaining high dose group animals, the cause of death was considered not to be substance related. The lung of this animal was sent to histopathology in order to exclude misdosing as possible cause of death. Behavior and state of health in all other animals remained uneffected by the administration of the test substance in all dose groups. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed.

Neurotoxicological examinations:
Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, and forelimb and hindlimb grip strength was not influenced by the administration of the test substance in any group.

Body weight:
Mean body weight was statistically significantly decreased in high dose males between study 5 and 29 d (ca. 10%, treatment period) and during recovery, when compared to the control. Mean overall body weight gain was reduced, accordingly. Body weight development was not influenced by administration of the test substance in high dose females, or in any other mid- and low dose group animals.

Food consumption:
Food consumption remained unaffected by the administration of the test substance throughout the study in all dose groups.

Clinical pathology

Hematology:
There were no changes in final hematology in any group, which could be related to administration of the test substance. Platelets were slightly but statistically significantly increased in high dose males. This finding, which was slightly outside the range of the historical control data for this rat strain and age, was not seen for high dose females, and hence, considered to be of no toxicological significance. Hematology following recovery was completely unobstrusive when compared to the respective control

Clinical chemistry:
There were no changes in clinical chemistry in any group that could be related to administration of the test substance. Statistically significant findings after final or recovery evaluation were observed sporadically, were only marginal and generally within the range of the historical control data for this rat strain and age(except for urea nitrogen and ALP in recovery males). Moreover, they were confined to one sex only, and hence considered not to be toxicologically significant.

Urinalysis:
Urinary function was not adversely influenced by administration of the test substance in any group. The slightly but statistically significant increase in the urinary specific weight in high dose males was very slight and within the range of biological variation and hence considered to be of no toxicological relevance.

ANATOMIC PATHOLOGY

Organ weights:
Final organ weights were not affected by administration of the test substance in any group. There was a statistically significant decrease (p<0.05) of absolute kidney and liver weight in recovery high dose males against the control. This finding correlated well with the lower terminal body weight and hence was considered not to be substance related.

Relative organ weights:
Accordingly, organ weights relative to body weight were not influenced by administration of the test substance in any group.

Macroscopic observations:
Relevant macroscopic changes could not be seen in male or female animals in all investigated groups.
One high dose female was killed early on 6 d of the experiment. Necropsy of this animal did not reveal macroscopic findings, hence it was not assigned for histopathological preparation with the exception of the lungs in order to exclude misdosing. Histopathological examination of the lungs did not reveal any pathological findings. Hence, death of this animal remains unclear. Stress during dosing procedure was considered as a possible cause of death. One further animal of dose group 1 (control, scheduled kill) exhibited a yellowish discoloured papillary processus of the liver.

Microscopic observations:
Stomach: mixed-cellular infiltrations in the following animals of terminal sacrifice groups: all animals with an average grading of 3.2 (male)/3.5 (female) in high dose group, three male and one female rat (average grading of 2.7/2.0) in mid dose group, compared to one animal (2.0) in the control group. The recovery group exhibited no comparable finding. Further sporadic findings in form of cellular infiltrations were observed in various organs of different animals; tubulardilatation in the kidney, hemorrhage in the urinary bladder, sperm granuloma, and hydrometra.

All these sporadic findings in the high dose group could be observed in the control group, too, or commonly occur in historical controls. These findings spontaneously observed in different organs of single animals of different groups should be interpreted as incidental findings.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the fact that administration of the test substance was tolerated without any adverse systemic effects, the NOAEL was considered to be the highest dose level of 800 mg/kg bw/day.

Executive summary:

A 28 d oral (gavage) repeated dose toxicity study was conducted in rat according to OECD Guideline 407, EPA: OPPTS 870.3050 and EU Method B.7 in compliance with GLP. Groups of male and female rats received the test substance at dose levels of 0, 50, 200, or 800 mg/kg bw/day by oral gavage for a period of 28 d. 14 d recovery groups (controls and high dose animals) were also included in the study. Except for one incidental death, there was no mortality and/or clinical findings throughout the study. Further, body weight development was not influenced by administration of the test substance in high dose females, or in any other mid and low dose group animals. Statistically significantly decreased mean body weight gains were observed in males at highest dose level, which was considered to be an incidental effect. In addition, statistically significantly lower mean absolute kidney and liver weights in high dose recovery males resulting from the lower terminal body weight in this group. No other organ weight changes were observed at any of the tested dose levels or in females. Further, no adverse effect of test substance was observed on behaviour, state of health, neurotoxicological parameters and food consumption at any of the tested dose level. Likewise, no test substance related alterations were observed on urinalysis, haematological and clinical chemistry parameters. Moreover, no substance related macroscopic findings was observed at necropsy. There were signs of local irritations in the stomach (mixed-cellular infiltrations) in particular in high dose group males and females which was clearly reversible throughout the post treatment period. This finding is due to oral gavage of the test substance and not relevant for humans. Based on the fact that administration of the test substance was tolerated without any adverse systemic effects, the NOAEL was considered to be the highest dose level of 800 mg/kg bw/day.