Reaction mass of copper complex of [(2,6-difluoroheterocycl-4-yl)amino]hydroxy{[2-hydroxy-3-sulfonato-5-(vinylsulfonyl)phenyl]diazenyl}naphthalene sulfonic acid, dialkali salt and copper complex of [(2,6-difluoroheterocycl-4-yl)amino]-hydroxy{[2-hydroxy-3-sulfonato-5-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl]diazenyl}naphthalene sulfonic acid, trialkali salt

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:

2003-04-14 to 2003-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3050

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Reaktiv Rot F00-0124

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 (e.g. ad libitum): ssniff R/M-H (V 1534) ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): Tap water in plastic bottles ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: at least five days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

First and last week of treatment phase

Duration of treatment / exposure:

28

Frequency of treatment:

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:

Male: 10 animals at 0 mg/kg bw/day (5 main group, 5 recovery group)
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 200 mg/kg bw/day
Male: 10 animals at 800 mg/kg bw/day (5 main group, 5 recovery group)
Female: 10 animals at 0 mg/kg bw/day (5 main group, 5 recovery group)
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 200 mg/kg bw/day
Female: 10 animals at 800 mg/kg bw/day (5 main group, 5 recovery group)

Control animals:

yes

Details on study design:

- Dose selection rationale:
An acute oral toxicity testing of Reaktiv Rot F00-0124 determined the median LD50 is above 2000 mg/kg bw in male and female rats. A dose range finding study in 3 male and 3 female rats receiving Reaktiv Rot F00-0124 at doses of 400 and 800 mg/kg bw/day over 14 days was conducted. Neither deaths nor clinical symptoms occurred. Based on these results, dose levels of 0, 50, 200 and 800 mg/kg body weight per day were selected for the present study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Survival control of the animals was examined twice daily (on weekends and public holidays once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were observed once daily

BODY WEIGHT: Yes
- Time schedule for examinations: determinated before the start of the study and then twice weekly throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes - Food consumption was determined continuously (two times per week)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes - Ketamine-Hydrochloride + Xylazine
- Animals fasted: No
- How many animals: al: Erythrocyte counts, Heinz Body Counts, Hematocrit, Hemoglobin, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Mean corpuscular volume, Reticulocyte counts, Differential leukocyte counts, Leukocyte counts, Coagulation time, Thrombocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After blood sampling for hematological testing, the animals were killed by section of the vena cava cranialis in deep narcosis and exsanguinated
- Animals fasted: No
- How many animals: all animals
- Parameters checked: gamma-Glutamyltranspeptidase, Alanine Aminotransferase, Albumin, Albumin / Globulin ratio, Alkaline Phosphatase, Aspartate Aminotransferase, Bilirubin total, Calcium, Chloride Cholesterol, Creatinine, Globulin, Glucose, Inorganic Phosphorous, Potassium, Sodium, Total Protein, Triglycerides, Urea, Uric Acid

URINALYSIS: Yes
- Time schedule for collection of urine: a few days before termination of the study
- urine was collected in diuresis cages (overnight from day 22 to day 23)
- Animals fasted: Yes
- Parameters checked: Appearance, Bilirubin, Blood, Color, Glucose, Ketone bodies, Microscopic Examination, pH, Protein, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first treatment and weekly thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena
- Battery of functions tested: 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 an 16-station automated motor activity monitoring device. 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- 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
- Endotracheal fixation of the lungs, Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus
HISTOPATHOLOGY: Yes
- Histopathological examinations were carried out of the control and high dose animals
- Adrenals, Bone marrow / sternum, Brain with medulla oblongata, Epididymides, Heart, Small Intestine jejunum, Large Intestine colon, Kidneys, Liver, Lungs, Lymph nodes mandibular, Lymph nodes iliac, Nerve sciatic nerve, Ovaries, Prostate, Seminal vesicle, Spinal cord cervical, Spleen, Stomach, Testes, Thymus, Thyroid gland with parathyroids, Trachea, Urinary bladder, Uterus, All other gross lesions

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Control and low dose group showed no clinical observations.
Animals from the mid dose group showed black discolored feces from day 13 up to the end of the treatment period (study day 29). One female animal of the mid dose group showed stilted gait and squatting posture from day 23 up to day 29, another female animal of this group showed squatting posture from day 27 up to study day 29 (end of treatment period). Due to the fact that only two animals of this dose group showed this finding and in the absence of any other relevant alterations in the mid dose group, these alterations are not considered as adverse findings as the health status is not affected.
Animals in the high dose group high dose showed black discolored feces from day 2 up to the end of the treatment period. Seven of ten males showed squatting posture and/or stilted gait from day 26/27 up to the end of the treatment period. These symptoms were also noted in all females of the high dose group starting in the last study week and lasting up to the end of treatment or even day 31 (three females of the recovery group).

Mortality:

no mortality observed

Description (incidence):

No deaths occurred throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains of the females treated with 800 mg/kg bw. per day (high dose) decreased within the treatment period (mean decrease day 1 to 29 : 14 %), while no alterations were noted in the other groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight analysis revealed no relevant alterations in all dose groups except of slightly decreased absolute liver weights in female animals treated with 800 mg/kg per day after the treatment period, which normalized after the recovery period.
No relevant changes in organ weights were observed in all dose groups after the treatment and recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals from the low dose group showed no macroscopic alterations. Two males from the mid dose group showed small testes and epididymides, which is considered as incidental finding in the absence of any macroscopic or microscopic observations in these organs in the high dose group.
In the high dose group three females showed dark brown discolored kidneys and two of them additionally black spleens after the treatment period. Black discolored spleens were also observed in three females of the high dose group after the recovery period.This is due to the staining effect of the dye

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical observations:
Control and low dose group showed no clinical observations. Animals of the mid dose group showed black discolored feces from day 13 up to the end of the treatment period. One female of the mid dose group showed stilted gait and squatting posturefrom day 23 up tp day 29, another female
from this dose group showed squatting posture from day 27 up to study day 29. Animals in the high dose group showed black discolored feces from day 2 up to the end of the treatment period. Seven of ten males showed squatting posture and/or stilted gait from day 26/27 up to the end of the treatment period. These symptoms were also noted in all females of the high dose group starting i the last study week and lasting up to day 29 or 31. No other symptoms and no opacitiy of the refracting media of the eyes,changes of the oral mucosa, or impairments of dental growth were observed in all groups. Neurotoxicological measurements showed no substance related effect.Body weight gains were not influenced by the administration of the test compound in the low and mid dose group and in males of the high dose group. Female animals of the high dose group showed a slightly reduced body weight gain during the treatment period. Food consumption remained uneffected by the administration of the test compound throughout the study in all dose groups.

Body weight and weight gain:
Body weight gains were not influenced by the administration of the test compound in the low and mid dose group and in males of the high dose group. Female animals of the high dose group showed slightly reduced body weight gains (mean reduction in body weight gain between day 1 and 29: 14 %) during the treatment period.

Effects in organs:
Organ weight analysis revealed no relevant alterations in all dose group except of slightly decreased absolute liver wights in female animals treated with 800 mg/kg per day after the treatment period which normalized after the recovery period. Animals from the low dose group showed no macroscopic alterations. In the high dose group three females showed dark brown discolored kidneys and two of them additionally black spleens after the treatment period. Black discolored spleens were also observed in three females of the high dose group after the recovery period. No test compound related histopatological findings were observed.

Gross pathology:
Animals from the low dose group showed no macroscopic alterations. Two males from the mid dose group showed small testes and epididymides, which is considered as incidental finding in the absence of any macroscopic or microscopic observations in these organs in the high dose group. In the high dose group three females showed dark brown discolored kidneys and two of them additionally black spleens after the treatment period. Black discolored spleens were also observed in three females of the high dose group after the recovery period.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

3

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Mean body weight gains [g] throughout the study

 

Treatment group   [mg/kg bw]	Control		50 mg/kg		200 mg/kg		800 mg/kg
	males	females	males	femals	males	females	males	females
day 1 to day 29	121.2	76.0	127.6	76.4	115.6	73.6	132.5	65.4
day 29 to day 43	28.6	12.6	-	-	-	-	32.0	20.0

 

Female animals of the high dose group showed slightly reduced body weight gains (mean reduction in body weight gain between day 1 and 29: 14 %) during the treatment period.

 

Mean food consumption [g/kg bw/day] throughout the study

 

Treatment group   [mg/kg bw ]	Control		50 mg/kg		200 mg/kg		800 mg/kg
	males	females	males	females	males	females	males	females
day 1 to day 29	87.95	88.97	88.28	88.69	88.11	90.17	87.94	88.53
day 29 to day 43	67.59	70.59	-	-	-	-	66.54	75.03

 

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

 

Changes in absolute organ weights (final value day 29)

Parameter	1	2	3	4	1	2	3	4
Liver	9.884	10.062	9.178	10.776	7.308	7.882	7.688	6.182-

 

Organ weight analysis revealed no relevant alterations in all dose groups except of slightly decreased absolute liver weights in female animals treated with 800 mg/kg per day after the treatment period, which normalized after the recovery period.

 

Changes in relative organ weights (final value day 29)

Parameter	1	2	3	4	1	2	3	4
Spleen (% of bw.)	0.24725	0.26654	0.26039	0.26453	0.29630	0.32113+	0.33153+	0.35142+

 

Relative liver weights were slightly but not but statistically significantly decreased after the treatment period in females of the high dose group, but the mean value (3.2765) correspond to the control value after the recovery period (3.2828). Additionally, relative spleen weights were slightly increased in females, but exceeded the historical control data range only marginally in the high dose group after the treatment period. In the low dose group none of the individual values was outside the historical range, while in the mid dose group only one female (No. 46) showed a relative spleen weight slightly outside the control range. Therefore alterations in relative spleen weights in the low and mid dose group are not considered as relevant or substance related findings. No significant alterations were observed after the recovery period.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, repeated administration of Reaktiv Rot F00-0124 at dose levels of 50 or 200 mg/kg/d did not cause any adverse substance related alterations.

Executive summary:

In a subacute toxicity study, Reactive Red F00-0124 was administered to 5 Sprague Dawley rats/sex/dose, with an additional 5/sex for the recovery group in the control and high dose group. The test substance was administered via oral gavage in deionized water at dose levels of 0, 50, 200 and 800 mg/kg bw/day.

The test item was well tolerated and resulted in no deaths or severe clinical symptoms. Neurotoxicological examination, hematology and clinical chemistry values as well as urinalysis and histopathological investigation showed no substance related or biologically relevant alterations in all dose groups.

Animals in the high dose group (800 mg/(kg/d) showed squatting posture and/or stilted gait at the end of the treatment period. Female animals of the high dose group showed additionally slightly reduced body weight gains during the treatment period (14 %), slightly decreased absolute liver weights. However, these changes were reversible and normalized after the recovery period. The NOAEL is 200 mg/kg bw/day, the LOAEL is 800 mg/kg/day. 

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407) in rats.