Tetrasodium 4-amino-5-hydroxy-6-[[2-methoxy-5-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-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:

From Apr. 19, 2005 to May 31, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

464-700-1

EC Name:

-

Cas Number:

607724-42-5

Molecular formula:

Hill formula: C28H24N5Na5O23S7 CAS formula: C28H29N5O23S7.5Na

IUPAC Name:

Pentasodium 4-hydroxy-3-(2-methoxy-5-(2-sulfooxyethanesulfonyl)phenylazo)-7-(sulfomethylamino)-8-(2-sulfo-4-(2-sulfooxyethanesulfonyl)phenylazo)naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

see below

Test animals

Species:

rat

Strain:

Sprague-Dawley

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: ssniff® R/M-H (V 1534) 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
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 50 ± 20 %
- Photoperiod: 12 h light / dark cycle

IN-LIFE DATES: From: Apr. 19, 2005 To: May 31, 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in the stated concentrations in deionized water. After each measurement of the body weight, the calculation of the application volume was recalculated and adapted

VEHICLE
- Concentration in vehicle: 0.0, 12.5, 50.0 and 200.0 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Frequency of administrations: Once daily

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

per HPLC in first and last week of study from samples of each concentration

Duration of treatment / exposure:

28 d

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five/sex: 62.5 and 250 mg/kg bw/day
Ten/sex: 0 and 1,000 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 2,000 mg/kg in the rat (limit test) showed that the median lethal dose (LD50) is above 2,000 mg/kg bw in female animals. The dose of 2,000 mg/kg bw was tolerated by all the animals.
Based on these results, dose levels of 0, 62.5, 250 and 1,000 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 assignet to the different groups (i.e., 0, 62.5, 250 and 1,000 mg/kg bw/day).

Examinations

Observations and examinations performed and frequency:

General health condition and behavior (inclusive mortality):
Survival, health condition and behavior were examined twice daily (on weekends and public holidays once daily).

Clinical observations:
Individual clinical observations were performed 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.

At the termination of the study sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive) was evaluated including startle reflex (click response), response to approach with the finger to the nose of the animal, and righting reflex. The presence and absence of pupillary constriction was assessed using a pen flashlight directed into the eye. Assessments of motor function were performed including measurement of motor activity, and forelimb and hindlimb grip strength. The animals were evaluated for motor activity during a 60-minute period in a 16-station automated motor activity monitoring device (FMI, Föhr Medical Instruments GmbH). Activity counts were recorded by the interruption of photocells in 3 minute-intervals to give a total of 20 intervals. Fore- and hindlimb grip strength were measured by a strain gauge device (FMI, Föhr Medical Instruments GmbH) measured.

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 continuously (two times per week).

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))

Serum 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 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 as well as before the end of the recovery period. For this purpose, the urine was collected in diuresis cages (overnight from Day 24 to Day 25 and from Day 36 to Day 37).. Food and water 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) and quantitative parameters (i.e., volume and specific weight)

Sacrifice and 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 formalin (10%) solution using a needle inserted into the trachea. Following completion of the endotracheal fixation the lungs were fixed, together with the other organs, in formalin (10%) 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 in formalin (10 %) and processed for histopathological investigations. The tissues or organs examined were adrenals, bone marrow / sternum, brain with medulla oblongata, epididymides, heart, small intestine (ileum), large intestine (colon), kidneys, liver, lungs, lymph nodes (mandibular and iliac), nerve (sciatic nerve), ovaries, prostate, seminal vesicle, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid gland with parathyroids, trachea, urinary bladder, uterus and all other gross lesions. Histopathological examinations were carried out of the control and high dose animals, all animals which died early as well as gross lesions in all other groups. Samples of organs mentioned above were embedded by conventional histological technique in Paraplast. All organs were stained with hematoxylin-eosin.

Statistics:

Dosing and Recovery Phase data of body weight (from Day 1 of dosing, twice weekly) was analyzed for statistical significance (p ≤ 0.05) within each sex by a 1-way ANOVA with a two sided ordinal step-down trend test (see Tukey et al., 1985). Hematological parameters, serum chemistry parameters, urinalysis and organ weights were analyzed accordingly, if normally distributed, or by the Jonckheere trend test with corrections for ties (Jonckheere AR, 1954; Lin, F.O. and Haseman, J.K., 1975), if not normally distributed.
In case of insufficient sample size per group (N<3 for parameters normally distributed and N<4 for parameters not normally distributed), no statistical analysis was performed.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

(The animals of the high dose group showed dark red discolored feces and tray / bedding was red discolored from Day 21 up to Day 29)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

(urine discoloaration in some of the intermediate and high dose animals)

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

(Most animals of the high dose group (9/10) showed light red discolored skin at the end of the treatment period and all animals of this group showed dark red (9 animals) or light red (one female animal) discolored kidneys)

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

General health condition and behavior (inclusive mortality):
No death occured throughout the study. General health condition and behavior showed no test substance related alterations.

Clinical observation:
The animals of the high dose group showed dark red discolored feces and tray / bedding was red discolored from Day 21 up toDay 29. These discolorations were no longer observed in the recovery period. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairments of dental growth was observed.

Neurotoxicological examinations:
Neurotoxicological measurements including 'open field' observations, assessment of sensory function (included in raw data), as well as forelimb and hindlimb grip strength and motoractivity were not influenced by the administration of the test substance in all groups. The statistical effect in the number of movements in females of the high dose group was assessed to be an incidental finding without test substance relation, because no dose dependency occurred.

Body weight:
Body weight gains were not influenced by the administration of the test substance in all groups. Female animals of the mid and high dose group showed slightly lower weights on Day 1 and temporarily in the course of the study, but no dose-dependency occurred indicating that this observation was incidental and not test substance related. Body weight gain in control and high dose group animals was comparable.

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

Hematology:
Hematology showed no toxicologically relevant alterations in all dose groups. Male animals of the high dose group showed slightly lowered reticulocyte levels, which were however not outside the historical normal range and even above recovery control values. Additionally, neutrophil levels were lowered in this dose group which was considered to be not relevant in the absence of any correlated histopathological findings. Marginally less cells were unclassified in this dose group compared with the control.

Statistically significant alterations (increased platelet count, decreased monocyte count, decreased eosinophil count in high dose recovery males and decrease coagulation time in high dose recovery females) were observed at the end of the recovery period in animals of the high dose were not considered toxicologically relevant, because they were slight, not correlated to findings at Day 29 and not outside of the historical control data (if available).
2 male control recovery animals (and 1 male control final value animal) were not investigated for blood coagulation because of an oversight, but this deviation was considered to be not having any influence on the study outcome, because coagulation values in animals treated with the test item had no alterations and platelet values were normal.

Serum chemistry:
No adverse alterations were observed in serum chemistry. Some parameters showed statistically significant deviations, but not outside the historical normal range. Bilirubin was altered simultaneously in both sexes at the end of the treatment period in the high dose group but did not exceed the normal range. The increase was 1.4 fold of the control in male and and 1.3 fold in female animals, but was considered to be caused by test substance in serum because of the following reasons: the test substance showed strong absorption at the wavelength of bilirubin measurement (550 nm), no bilirubin was detected in urine at the end of the recovery period, apparent urinary bilirubin measurements were low, as no relevant increases in serum bilirubin levels were detected at the end of the treatment period and as no histopathological findings at all were present in the liver of these animals, a relevant test substance related hepatotoxic effect is excluded.

Triglycerides in male animals of this dose group were also increased (1.3-fold), but decreased in female animals (0.8-fold) and values were within the normal range. All other alterations described in the table were marginal (glucose, potassium) or without dose-dependency (sodium, chloride,
total protein, globulin, albumin/globulin ratio). GGT activity was below the limit of detection (2U/L) in most animals.

The recovery group animals showed some statistical significant alterations in serum chemistry parameters, which were within historical control range for these paramters, hence not considered adverse.

Urinalysis:
Urine was red discolored at the end of the treatment period in one female animal in the mid dose group and in 4 female as well as all male animals in the high dose group. The 5th female animal and most animals of the intermediate dose group showed dark yellow urine which was also considered to be indicative for urinary test substance elimination. At the end of the recovery period two male and one female animal still showed red urine discoloration. Red urine discoloration at the end of the treatment period correlated (with exception of one animal showing dark yellow urine) with small to moderate apparent bilirubine levels. At the end of the recovery period no bilirubin was detected in urine. Bilirubin measurements were considered to be false
positive due to test item absorption at the wavelength of bilirubin detection (550 nm). One male animal of the high dose group showed blood in urine at the end of the treatment period without any histopathological correlate. Hence, this finding was interpreted as isolated non test substance related finding.

ANATOMIC PATHOLOGY
Organ weights:
No toxicologically relevant alterations were observed in organ weight analysis. Some organ weights (liver and kidney weight in high dose males) showed statistically significant deviations, but most values were within the historical control data range. Only absolute and relative spleen weight in females of the high dose group were slightly above the historical range, but the absolute value corresponded largely to the control value of recovery animals and moreover, no histopathological correlate was observed, indicating that the finding was not relevant.

Macroscopic observations:
Most animals of the high dose group (4 males and 5 females) showed light red discolored skin at the end of the treatment period and all animals of this group showed dark red (9 animals) or light red (one female animal) discolored kidneys. At the end of the recovery period all male animals, but less females (3/5) of the high dose group still showed light red discolored skin. Kidney discolorations were no longer observed after the recovery period. No further test item related alterations were noted at necropsy. One male control animal showed hernia (ectopia) at the right accessory liver lobe.

Microscopic observations:
There were no histopathological findings, which could be related to the administration of the test substance in all dose groups. The discolorations of kidneys and skin at terminal necropsy were test article related, but no histopathological equivalent was detected. In the ectopic liver observed in male control animal, capsular fibrosis was detected.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 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:

Under the test conditions, repeated administration of the test substance at dose levels of 62.5, 250 and 1,000 mg/kg bw day did not cause any toxicologically relevant alteration. Hence, the NOAEL for the test substance was determined to be above 1000 mg/kg bw/day.

Executive summary:

A 28 d oral (gavage) repeated dose toxicity study was conducted in rats according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. Groups of male and female rats received the test substance at dose levels of 0, 62.5, 250 or 1,000 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. No unscheduled deaths were recorded throughout the study. Behaviour, state of health, body weight development, food consumption, neurotoxicological investigations, hematological analysis, serum chemistry analysis, organ weights measurements, urinalysis, necropsy and histopathology did not reveal any toxicologically relevant alterations. Test substance related discolorations occurred during the study. From Day 21 up to Day 29, the animals of the high dose group showed dark red discolored feces and tray / bedding was red discolored. These discolorations were no longer observed in the recovery period. Urine was red discolored at the end of the treatment period in one female animal in the mid dose group and in 4 females as well as all male animals in the high dose group, indicating test item elimination. At the end of the recovery period, two male and one female animal still showed red urine. This finding was not considered to be toxicologically relevant. Most animals of the high dose group (9/10) showed light red discolored skin at the end of the treatment period and all animals of this group showed dark red (9 animals) or light red (one female) discolored kidneys. At the end of the recovery period, all males but less females (3/5) of the high dose group still showed light red discolored skin. Kidney discolorations were no longer observed after the recovery period. No histopathological equivalent for the discolorations was detected. Under the test conditions, repeated administration of the test substance at dose levels of 62.5, 250 and 1,000 mg/kg bw/day did not cause any toxicologically relevant alteration. Hence, the NOAEL was determined to be 1,000 mg/kg bw/day.