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

Endpoint summary

Description of key information

No toxicological relevant adverse effect was seen in any of the short-term repeat dose studies with close structural analogues used for assessment. The NOAEL was therefore considered to be above 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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

Dose / conc.:

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

Dose / conc.:

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

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).

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.

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.

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

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20, 2002 to August 01, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: OPPTS 870.3050

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MITI: Guidelines for screening toxicity testing of chemicals

Deviations:

no

GLP compliance:

yes

Limit test:

no

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, 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
- Animal identification: Tatto number on the tail and cage numbering and for marking with potarsuim permanganerte (KMnO4)
- 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: June 20, 2002 to August 01, 2002

Route of administration:

oral: gavage

Vehicle:

other: Sesame oil

Details on oral exposure:

-PREPARATION OF DOSING SOLUTIONS: Test substance was suspended in the stated concentrations in sesame oil. After each measurement of the body weight, the calculation of the application volume was repeated.
- Concentration in sesame oil (vehicle): 12.5, 50.0 and 200.0 mg/mL
- 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:

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

Duration of treatment / exposure:

29 d, 28 applications

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

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 2000 mg/kg bw in both male and female animals. The dose of 2,000 mg/kg bw was tolerated by all the animals without signs of intoxication.
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 respective groups (i.e., 0, 62.5, 250 and 1,000 mg/kg bw/day).

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.

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. A strain gauge device (FMI, Föhr Medical Instruments GmbH) measured fore- and hindlimb grip strength.

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))

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 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, using metabolism cages (overnight from 26 d to 27 d and from 36 d to 3 d) collected the urine. 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)
* This parameter was only evaluated in the animals from the control and high dose group.

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 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, ovaries, 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 / femur, brain with medulla oblongata, epididymides, eyes with optic nerve, heart, small intestine 2 jejunum, large intestine 2 colon, kidneys, liver, lungs, lymph nodes 1 mandibular, lymph nodes 2 iliacal, nerve sciatic nerve, ovaries with oviducts, prostate, pituitary, 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. Histopathological examinations were carried out of the control and high dose 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

Clinical signs:

no effects observed

Description (incidence and severity):

High dose males and females exhibited discolored feces from 5 d onwards, which was related to test substance

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

clinical chemistry revealed signs of a mild metabolic overload for the high dose group (1000 mg/kg bw/day) primarily associated with disturbances of the serum lipid household (increased triglycerides, bilirubin, AP activity [males], albumin)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

statistically significantly increased specific urine weight for high dose males, and slight bilirubinuria for high dose males and females, which is the result of an interference of the dye with the photometric assay

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

hindlimb grip strength was statistically significantly decreased in high dose females, when compared to the control. However, this finding was not considered to be test substance related in the absence of any other neurobehavioral correlate

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were moderately increased for high dose males and females, attaining statistical significance only in the males. Kidney weights were slightly increased for high dose males and females, attaining statistical significance in females only.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

olive/dark brown discoloured kidneys in high dose males and females, as well as discoloured stomachs in two high dose males

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

Behavior, state of health and mortality
There were no unscheduled deaths throughout the study.
Clinical observation
Behavior and state of health 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. High dose males and females exhibited discolored feces from 5 d onwards, which was related to test substance.

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 all groups.
Hindlimb grip strength was statistically significantly decreased in high dose females, when compared to the control. This finding was not considered to be test substance-related in the absence of any other neurobehavioral correlate in this or the other gender of this group. Moreover, the value was within the physiological range of this sex strain and gender

Body weight
Body weight gains were not influenced by the administration of the test substance in all groups
Food consumption
Food consumption remained unaffected by the administration of the test substance throughout the study in all dose groups.

CLINICAL PATHOLOGY
Hematology
Final hematological evaluation as well as hematology after recovery did not reveal any test substance related changes in any group. Reticulocytes were slightly but statistically significantly increased in high dose males against the control. The value was well within the range of the historical control data for this rat strain and age. There was a statstically significant increase in the RBC counts for high dose recovery females, which was slightly outside the upper range of the historical control data. However, such finding was considered not biologically significant.
There was no evidence of an adaptive response of reticulocytes to an anemia, in the absence of any changes in the RBC picture. Hence, these minor differences from the controls were considered incidental.

Clinical chemistry
Final clinical chemistry evaluation revealed statistically significant increases, for high dose group animals, in total bilirubin, triglycerides and albumin. In addition, alkaline phosphatase activity was statistically significantly increased for high dose males and potassium levels increased for high dose females. All these findings were generally slight to moderate and at the upper physiological range for this rat strain and age.
The extraordinary marked increase in uric acid for the females could be traced back to one single animal, which exhibited a value of 530 umol/L, probably due to a false positive response of the serum sample. The slightly high uric acid level of high dose males was due to an extraordinary low value for the respective controls, being outside the lower range of the historical control data.
Triglycerides were still slightly increased for high dose males after the 14 d recovery period, but had completely reversed in high dose females.
All other changes of statistical significance after final- or recovery-evaluation were considered not toxicologically significant.

Urinalysis

Final urinalysis revealed statistically significantly increased specific urine weight for high dose males, and slight bilirubinuria for high dose males and females, when compared to the controls.
None of these findings were seen in urinalysis after the 14 d recovery period

ANATOMIC PATHOLOGY
Organ weights
Liver weights were moderately increased for high dose males and females, attaining statistical significance only in the males. Kidney weights were slightly increased for high dose males and females, attaining statistical significance in females only.
Liver weights were still increased for high dose recovery males, but had completely reversed in high dose females. Kidney weights had also returned to normal after the recovery period, in both genders.
Relative organ weights
Likewise, liver and kidney weights relative to terminal body weight were increased in high dose males and females, with clear tendency (liver, males) or complete recovery thereafter.
Macroscopic observations
Macroscopic evaluation at final necropsy revealed olive/dark brown discoloured kidneys in high dose males and females, as well as discoloured stomachs in two high dose males.
No other test substance-related macroscopic changes could be seen in male or female animals in all investigated groups. The livers of one mid dose male, and one high dose male, exhibited a beige patch in the right accessory lobe, which could be confirmed histopathologically as circumscriptive necrosis. This artefactual necrosis was considered to be due to intraperitoneal injection of the anaesthetic drug at final necropsy. A test substance-relationship could be excluded.

Microscopic observations
There were no histopathological findings in final group animals that could be related to administration of the test substance.

The following histopathological findings were recorded:
-Stomach: mixed-cellular infiltrations observed in the four males and four females with an average grading of 2.3 or 2.0, respectively in high dose group. Also, in the one male and four females with an average grading of 2.0, in control group.
-The histopathological equivalent to the beige patches in the liver was a lobular necrosis in one animal in high dose group and one animal in mid dose group.
-One animal in high dose group showed tubular dilatations in the kidneys, and one female in control group a hemorrhage in the thymus.
-Further sporadic findings in form of inflammatory foci / infiltrates were observed in heart, liver, lungs, prostate and uterus of single animals randomly distributes over all investigated groups. The other findings sporadically observed in various organs of animals from different groups occurred at an incidence and severity historically seen at the test facility for rats of their age and strain. Therefore, they were not considered related to treatment

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

-The cellular infiltration in the submucosal area of the glandular stomachs could be observed in several animals of all investigated groups; it should therefore be discussed as a topical slight acute but unspecific irritation, obviously not related to treatment

-Final clinical chemistry revealed signs of a mild metabolic overload for the high dose group (1000 mg/kg bw/day) primarily associated with disturbances of the serum lipid household (increased triglycerides, bilirubin,alkaline phosphatase(AP) activity [males], albumin). These clinical pathology changes were mild and, though associated with respective liver weight-increase (ca. 18%), did not show any histopathological correlate. Likewise, apart from pigment storage in the kidneys and slightly higher kidney weight, urinary function was not impaired in these animals, nor did it show a histopathological correlate.

 

Conclusions:

Final clinical chemistry revealed slightly increased triglycerides, bilirubin, AP activity [males], albumin which lie still within the historical control values. This increase could either be a sign of a mild metabolic overload for the high dose group (1000 mg/kg bw/day) as shown by slightly higher liver weights or due to interference of the photometric method (esp. bilibubin) by the discoloration of serum and urine resulting from the test substance absorption/excretion. As these clinical pathology changes were mild, did not show any histopathological correlate and were reversible within the 14-day recovery period, they were not considered to be adverse effects. Hence the NOAEL was considered to be 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, EPA: OPPTS 870.3050, MITI guideline and EU Method B.7 in compliance with GLP. Groups of male and female rats received the test item at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day by oral gavage for a period of 28 days. 14-day recovery groups (controls and high dose animals) were also included in the study.

There were no unscheduled deaths throughout the study. Behaviour, state of health, body weight gains and food consumption were not influenced by the administration of the test substance. High dose animals showed discoloured faeces from Day 5 onwards, which was caused by the colour of the test item. Neurotoxicology measurements and haematological evaluation did not reveal any test substance-related changes.

In clinical chemistry, slightly increased triglycerides, bilirubin, AP activity [males], albumin values were observed, which lie still within the historical control values. This increase could either be a sign of a mild metabolic overload for the high dose group (1000 mg/kg bw/day) as shown by slightly higher liver weights or due to interference of the photometric method (esp. bilibubin) by the discoloration of serum and urine resulting from the test substance absorption/excretion. Additionally, slightly higher kidney weights were observed in this dose group in both sexes at the end of the treatment period. Macroscopic evaluation at final necroscopy revealed that kidneys in the high dose group were discoloured by the test substance, as well as stomachs in two high dose males. These changes at 1000 mg/kg bw/day were fully reversible in females and showed tendency to recovery in the males, after the 14-day recovery period. No test substance-related histopathological findings were detected.

 

Based on alteration in clinical chemistry parameters and organ weight as well as pigment storage in the kidneys at 1000 mg/kg bw/day, the NOEL were established at the next lower dose of 250 mg/kg bw/day. As these were mild, did not show any histopathological correlate and were reversible within the 14-day recovery period, they were not considered to be adverse effects, hence the NOAEL was considered to be 1000 mg/kg bw/day.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1. Feb to 1 Mar 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Strain: Hoe:WISKf (SPF71)
- Age at study initiation: approx. 6 weeks
- Housing: 5 rats/cage
- Diet (ad libitum): Altromin 1324
- Water (ad libitum): tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Preparation: daily
- Concentration in vehicle: 1.25, 5, 20%
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Per HPLC in first and last week of treatment

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION: Yes
- Time schedule: twice per week
- Calculation: food consumption/100g bw/week

WATER CONSUMPTION : Yes
- Time schedule: once weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: No
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: overnight Day 26/27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes
HISTOPATHOLOGY: Yes

Statistics:

Yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

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):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Gross pathology: Reddish discoloration in kidneys and testes at 1000 mg/kg/day
Histopathology: In 2 females resorption vacuoles with dye in tubular epithelial cells of convoluted segment of the proximal tubulus of the renal cortex at 1000 mg/kg/day

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 1000 mg/kg: Discoloration of exctreta and organs. Renal re-absorption of dye - not considered to be toxicological relevant adverse effects.

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The oral administration of the test substanceat a dose of 1000 mg/kg body weight/day for 4 weeks resulted in microsopically visible re-absorption of the dye in the kidneys of two female animals. There was no evidence in clinical chemistry and histopathology of an adverse effect on the renal function. The administration of the test compound in a dose of 1000 mg/kg body weight/day did not induce any evident relevant compound-related changes in male animals. The NOAEL was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

The test substance was administered orally by gavage to SPF-Wistar rats over a period of 29 days (a total of 28 applications, 7 days a week) at dose levels of 0.62.5.250 or 1000 mg kg body weight. Behavior and general state of health were examined in all study groups. Body weights and food consumption were recorded twice a week, water consumption once weekly.

Hematology, clinical chemistry and urinalysis were performed at study end. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Relevant organs of the animals were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, albumin and globulin values, urine data (pH-value and specific weight) as well as the absolute and relative organ weights were analyzed with the aid of a statistical program to show differences compared to control groups.

 

Behavior, general state of health, body weight development as well as food and water consumption remained unaffected by the administration of the test compound. Feces of animals of the 1000 mg/kg test group were red discolored, beginning with day 6.

There was no evidence for compound-related toxicity in hematology and clinical chemistry. Urine was red-brown discolored in all animals of the 1000 mg/kg test group.

Evaluation of absolute and relative organ weights showed no compound-related effects.

Necropsy revealed red discoloration of the kidneys in all animals of the 1000 mg/kg test group. Additionally, the testes of the male animals were red discolored. Vacuolization of the epithelial cells of the renal cortex was microscopically observed in two female animals of this test group which are considered to be a result of an increased re-absorption and subsequent deposition of the test compound.

Summarizing, the 29-day oral administration of the test substance at a dose of 1000 mg/kg body weight/day resulted in two female animals in microsopic renal findings as described above. There was no evidence in clinical chemistry and histopathology of an adverse effect on the renal function. The administration of the test compound in a dose of 1000 mg/kg body weight/day did not induce any evident relevant compound-related changes in male animals. The NOAEL was therefore considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality database.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 21-day study with 14 applications of the structural analogue Reactive Black 5 was performed with a single dose of 500 mg/kg bw/day was performed without revealing any adverse effects. The only effects seen were substance stained urine and faeces.

 

Structural Analogue 01:The test substance was administered orally by gavage to SPF-Wistar rats over a period of 29 days (a total of 28 applications, 7 days a week) at dose levels of 0.62.5.250 or 1000 mg kg body weight. Behaviour and general state of health were examined in all study groups. Body weights and food consumption were recorded twice a week, water consumption once weekly. Haematology, clinical chemistry and urinalysis were performed at study end. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Relevant organs of the animals were processed for histopathological examination and checked for microscopically visible changes. Body weights, haematological and clinical chemistry data, albumin and globulin values, urine data (pH-value and specific weight) as well as the absolute and relative organ weights were analysed with the aid of a statistical program to show differences compared to control groups.

Behaviour, general state of health, body weight development as well as food and water consumption remained unaffected by the administration of the test compound. Faeces of animals of the 1000 mg/kg test group were red discoloured, beginning with day 6.There was no evidence for compound-related toxicity in haematology and clinical chemistry. Urine was red-brown discoloured in all animals of the 1000 mg/kg test group. Evaluation of absolute and relative organ weights showed no compound-related effects.

Necropsy revealed red discoloration of the kidneys in all animals of the 1000 mg/kg test group. Additionally, the testes of the male animals were red discoloured. Vacuolization of the epithelial cells of the renal cortex was microscopically observed in two female animals of this test group which are considered to be a result of an increased re-absorption and subsequent deposition of the test compound. However, no adverse tissue reaction resulted from these deposits and no correlating changes in clinical pathology was observed, hence these deposits were of no toxicological relevance.

Summarizing, the 29-day oral administration of the test substance at a dose of 1000 mg/kg body weight/day resulted in two female animals in microscopic renal findings as described above. There was no evidence in clinical chemistry and histopathology of an adverse effect on the renal function. The administration of the test compound in a dose of 1000 mg/kg body weight/day did not induce any evident relevant compound-related changes in male animals. The NOAEL was therefore considered to be 1000 mg/kg bw/day.

 

Structural Analogue 02: A 28 d oral (gavage) repeated dose toxicity study was conducted in rats according to OECD Guideline 407, EPA: OPPTS 870.3050, MITI guideline and EU Method B.7 in compliance with GLP. Groups of male and female rats received the test item at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day by oral gavage for a period of 28 days. 14-day recovery groups (controls and high dose animals) were also included in the study.

There were no unscheduled deaths throughout the study. Behaviour, state of health, body weight gains and food consumption were not influenced by the administration of the test substance. High dose animals showed discoloured faeces from Day 5 onwards, which was caused by the colour of the test item. Neurotoxicology measurements and haematological evaluation did not reveal any test substance-related changes.

In clinical chemistry, slightly increased triglycerides, bilirubin, AP activity [males], albumin values were observed, which lie still within the historical control values. This increase could either be a sign of a mild metabolic overload for the high dose group (1000 mg/kg bw/day) as shown by slightly higher liver weights or due to interference of the photometric method (esp. bilibubin) by the discoloration of serum and urine resulting from the test substance absorption/excretion. Additionally, slightly higher kidney weights were observed in this dose group in both sexes at the end of the treatment period. Macroscopic evaluation at final necroscopy revealed that kidneys in the high dose group were discoloured by the test substance, as well as stomachs in two high dose males. These changes at 1000 mg/kg bw/day were fully reversible in females and showed tendency to recovery in the males, after the 14-day recovery period. No test substance-related histopathological findings were detected.

Based on alteration in clinical chemistry parameters and organ weight as well as pigment storage in the kidneys at 1000 mg/kg bw/day, the NOEL were established at the next lower dose of 250 mg/kg bw/day. As these were mild, did not show any histopathological correlate and were reversible within the 14-day recovery period, they were not considered to be adverse effects, hence the NOAEL was considered to be 1000 mg/kg bw/day.

 

Structural Analogue 03: 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, haematological 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 discoloured faeces and tray / bedding was red discoloured. These discolorations were no longer observed in the recovery period. Urine was red discoloured 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 discoloured skin at the end of the treatment period and all animals of this group showed dark red (9 animals) or light red (one female) discoloured kidneys. At the end of the recovery period, all males but less females (3/5) of the high dose group still showed light red discoloured 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 1000 mg/kg bw/day did not cause any toxicologically relevant alteration. Hence, the NOAEL was determined to be 1000 mg/kg bw/day.

 

Based on this data from structural analogues, it can be concluded that the NOAEL for Reactive Blue 250 is also 1000 mg/kg bw/day.

Justification for classification or non-classification

Oral route

Based on the results of an oral repeated dose toxicity study, the test substance does not need to be classified for repeated dose toxicity according to the EU CLP criteria (EC 1272/2008) and EU Directive 67/548/EEC.

Dermal route

No study available.

Inhalation route

No study available.