REAKTIV-ORANGE DYPR 1410

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:

09 November 1998 to 21 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

432-080-1

EC Name:

-

Molecular formula:

Hill formula: C18H15N3Na2O9S3 CAS formula: C18H17N3Na2O9S3.2Na

IUPAC Name:

Disodium 4-amino-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

microgranulate or well dedusted powder

Details on test material:

Name: Reaktiv-Orange DYPR 1410

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Sprague Dawley rat
Strain: HSD: Sprague Dawley SD
Origin: HARLAN WINKELMANN Gartenstr. 27, 33178 Borchen SPF breeding colony
Age at the start of the study: approximately 6 weeks
Animal maintenance: in fully air-conditioned rooms in makrolon cages (type 4) on soft wood granulate in groups of 5 animals
Room temperature: 22 ±3 °C
Relative humidity: 50 ± 20 %
Lighting time: 12 hours daily
Acclimatization: at least five days
Food: 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 diu¬resis cages
Animal identification: animal tattooing and cage numbering
Randomization: computer-generated algorithm

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionised

Details on oral exposure:

Dose Concentration Volume applied Vehicle
(mg/kg b.w.) in % (w/v) (ml/kg b.w.)

0.0 0.00 5 deionised water
62.5 1.25 5 deionised water
250.0 5.00 5 deionised water
1000.0 20 00 5 deionised water

Reaktiv-Orange DYPR 1410 was dissolved in the stated concentrations in deionized water at the following dates:
1. November 09, 1998
2. November 23, 1998
After each measurement of the body weight, the calculation of the application volume was repeated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Week 1 and Week 4
HPLC

Duration of treatment / exposure:

29 days, 28 applications

Frequency of treatment:

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:

Group Dose (mg/kg b.w.) Number of animals Animal number
male female male female

1 0.0 10 10 1 -10 31 -40
2 62.5 5 5 11 -15 41 -45
3 250.0 5 5 16-20 46-50
4 1000.0 10 10 21 -30 51 -60

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for species selection: The rat has proved to be a suitable species for subacute oral toxicity testing with many different substances and is the species of choice according to the international guidelines.
Rationale for the route of exposure: The oral route is considered to be a potential exposure route in man.
Rationale for dose-selection; Acute oral toxicity testing of Reaktiv-Orange DYPR 1410 in the rat yielded a median lethal dose (LD50) above 2000 mg/kg body weight in both male and female animals. The dose of 2000 mg/kg was tolerated by all the animals without any signs of intoxication.
Based on this result, Reaktiv-Orange DYPR 1410 was tested in the present study at the dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day.

Groups of male and female rats received Reaktiv-Orange DYPR 1410 by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for a period of 28 days. On day 29 five males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 days.

Examinations

Observations and examinations performed and frequency:

Behavior and state of health
The behavior and general health condition of the animals were observed once daily.

Neurotoxicological examinations
Once before the first treatment and thereafter once a week detailed clinical observa¬tions 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 iacrimation, 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 flash¬light 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 (FMI. Fohr Medical Instruments GmbH). Activ¬ity 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, Fbhr Medical Instruments GmbH).

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

Food and water consumption
Food consumption was determined continuously (2 times per week). The values on the printouts refer to the intervals between one measurement and the next, They are converted to the food consumption per 100 g body weight over a 24 hour period,
Water consumption was determined once weekly over a period of 16 hours (from approx, 3.15 p.m. to 7.15 a.m.)

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 body weight Ketamine-Hydrochloride + Xylazine). In order to prevent systematic errors, blood sampling was conducted in a randomized order. Table of paramters determined listed under any other information.

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. Table of paramters determined listed under any other information.

Urine analysis
Urine analysis was performed on alt animals a few days before termination of the study. For this purpose, the urine was collected by using metabolism cages (overnight from day 25 to day 26). Food and water were withdrawn during this period.
Additionally, appearance and color of the urine were investigated a few days before the end of the recovery period (from day 39 to day 40). Table of paramters determined listed under any other information.

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 endotracheal with a formaldehyde 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 in formaldehyde solution together with the other organs.

Organ weights
The following organs were weighed and the organ to body weight ratios calculated:
Heart, Testes, Liver, Epididymides, Kidneys, Thymus, Adrenal glands, Brain & Spleen

Histopathology
The following tissues or organs (or pieces of them) were preserved in a suitable fixative and processed for histopathological investigations:
Heart, Brain, Lungs, Thymus, Liver, Trachea, Spleen, Thyroid glands with parathyroid glands, Kidneys, Testes, Stomach, Epididymides, Jejunum, Ovaries, Colon, Uterus, Urinary bladder, Lymph nodes iliac, Lymph nodes (mandibular), Adrenal glands, Prostate gland, Bone marrow (sternum), N.ischiadicus, Spinal cord (cervical)

Other examinations:

None

Statistics:

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

slightly increased leukocyte counts in females from the high dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

slight increases in total bilirubin and protein values in males from the high dose group

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

discolored salmon pink in males from the high dose group

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Behaviour, state of health and mortality
No deaths occurred throughout the study. Behaviour and state of health remained unaffected by the administration of the test compound. One animal from the high dose group (#25} did not show palpable testes from day 22 to the end of the study. At necropsy this animal was diagnosed as a hermaphrodite, as ovary and uterus (right side) as well as testes and epididymis (left side) were present. Therefore, the values from this animal were excluded from further analyses.

Opacity of the refracting media of the eyes and changes of the oral mucosa were not observed.


Neurotoxicological examinations
Neurotoxicological measurements including open field observations, assessment of sensory function, motor activity, forelimb and hindlimb grip strength did not indicate any compound-related effect

Body weight gain
Body weight gains remained unaffected by the administration of the test compound in all groups

Food and water consumption
Food consumption remained unaffected by the administration of the test compound throughout the study.
Food consumption was slightly increased in males from the high dose group. However, these changes were minor and occurred in one sex only. Therefore, a compound-related effect is questionable.
Likewise, the administration of the test compound did not alter the water consumption of the animals.
Water consumption was slightly increased in males of the high dose group during the whole observation time. Since this change was present before treatment commenced, a compound-related effect is questionable.

Hematological investigations
Final value: Statistical evaluation revealed decreases in red blood cell counts and reticulocyte counts in males from the high dose group. However, even lower values were observed in recent control males. Therefore, a compound-related effect is questionable.
Leukocyte counts were slightly, but statistically significantly increased in females from the high dose group.
Recovery value: No statistically significant changes were observed.
Heinz bodies were not detected. Likewise, differential white cell count did not reveal any abnormalities.

Clinical chemistry
Final value: Total bilirubin, protein and albumin values were statistically significantly increased in males from the high dose group. Protein and albumin values were also increased to a statistically significant degree in males from the intermediate dose group, but without dose-dependency. Therefore, a compound-related effect is unlikely. The increase in bilirubin is very common and well known for reactive dyes, as they can usually cause a slight staining of the serum when dosed. As bilirubin is measured photometrically such observations are often made.
Recovery value: Statistical evaluation revealed increases in Cholesterol levels (males) and direct bilirubin values (females). As normal values occurred at the end of the treatment period, a compound-related effect is unlikely.

Urine analysis
Final value: Urine was discoloured salmon pink in males from the high dose group. The sediments were inconspicuous.
Recovery value: No treatment-related changes were detected at urine analysis by the end of the recovery period.

Organ weights
Final value: Statistical evaluation revealed decreased brain weights In females from the high dose group. This change is probably due to the higher body weights of these animals.
Recovery value: Testes weights were increased to a statistically significant degree in males from the high dose group, probably due to the lower body weights of these animals.

Macroscopic and microscopic findings
No compound-related macroscopically visible changes were observed at necropsy. Likewise, histopathological examinations did no reveal any compound-related effect.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

serum/plasma biochemistry

urinalysis

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

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:

With regard to the results of the presented subacute toxicity study, the 'No Observed Effect Level' (NOEL) is 250 mg/kg body weight per day in males and females. However, as no adverse effects were observed at the dose level of 1000 mg/kg body weight per day, this dose level is considered as NOAEL.

Executive summary:

The present 29-day toxicity study was conducted in order to characterize the toxicological profile of Reaktiv-Orange DYPR 1410 after repeated oral exposure. Additionally, the results of this study can be used as a dose-range finding for subchronic and chronic toxicity studies.

The study was conducted in compliance with EEC-Guideline B.7."Subacute Oral Toxicity" of the Directive 96/547EEC and OECD Guidelines for Testing of Chemicals, 407 “Repeated Dose 28-day Oral Toxicity Study in Rodent". This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

Groups of male and female rats received Reaktiv-Orange DYPR 1410 by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for a period of 28 days. On day 29 five males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 days.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly. 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'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth. Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Haematological examinations and clinical chemistry were carried out at the end of the treatment period and after the recovery period. Urine analysis was performed at the end of the treatment period and after the recovery period. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, haematological and clinical chemistry data, urine data (volume, specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analysed with the aid of a statistical program to show differences compared to the controls.

No deaths occurred throughout the study. Behaviour and state of health remained unaffected by the administration of the test compound in all dose groups. Neurotoxicological parameters were comparable in all groups.

Body weight development, food and water consumption remained unaffected by the administration of the test compound in all groups.

Haematological examinations revealed slightly increased leukocyte counts in females from the high dose group. Clinical chemistry examinations revealed slight increases in total bilirubin and protein values in males from the high dose group. These changes were no longer observed at the end of the recovery period.

Urine was discoloured salmon pink in males from the high dose group. This change was no longer observed at the end of the recovery period.

Organ weights were not affected by the administration of the test compound. No compound-related macroscopically visible changes were observed at necropsy. Likewise, histopathological examinations did not reveal any compound-related effect.

In conclusion slight increases in bilirubin and protein levels in males and slightly increased leukocyte counts in females were observed after oral administration of Reaktiv-Orange DYPR 1410 (28 times during 29 days) at the dose level of 1000 mg/kg body weight per day. However, there were neither histopathological changes nor evidence for an impairment of liver function. The increase in bilirubin is very common and well known for reactive dyes, as they can usually cause a slight staining of the serum when dosed. As bilirubin is measured photometrically such observations are often made. Therefore, the above-mentioned findings are considered not to be of toxicological relevance. Additionally, salmon pink discolored urine occurred in males at this dose level, probably due to the colour of the test compound and/or its metabolites.
No compound-related effect was observed after repeated administration of Reaktiv-Orange DYPR 1410 at the daily dose levels of 62.5 and 250 mg/kg body weight/day in both sexes.
With regard to this result, the 'No Observed Effect Level' (NOEL) is 250 mg/kg body weight per day in males and females. However, as no adverse effects were observed at the dose level of 1000 mg/kg body weight per day, this dose level is considered as NOAEL.