Sodium 3-[(1,5-dihydroxy-2-naphthyl)azo]-4-hydroxybenzenesulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1999 to 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test compound: Diamantechtrot BT

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, Gartenstr. 27, 33178 Borchen, Germany, SPF breeding colony
- Age at study initiation: approximately 6 weeks
- Housing: in makrolon cages (type 4) on soft wood granulate in groups of 5 animals
- 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 %
- Air changes (per hr): ca 16
- Photoperiod (hrs dark / hrs light): 12 hours daily

IN-LIFE DATES: From: September 27, 1999 To: November 08, 1999

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dissolved in deionized water every 14 days

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA
- Concentration in vehicle: 1.5, 5, 20 % (w/v)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): -
- Purity: -

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to study start, the stability and homogeneity of the test substance in vehicle was tested at 1 and 250 mg/mL

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
62.5, 250, 1000 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
12.5, 50, 200 mg/mL
Basis:
nominal in water

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day Male: 5 animals at 62.5 mg/kg bw/day Male: 5 animals at 250 mg/kg bw/day Male: 10 animals at 1000 mg/kg bw/day Female: 10 animals at 0 mg/kg bw/day Female: 5 animals at 62.5 mg/kg bw/day Female: 5 animals at 250 mg/kg bw/day Female: 10 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): randomized
- Rationale for selecting satellite groups: to examine reversibility of effects seen
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): randomized

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Behavior and general health condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes: intraperitoneal injection of 67 + 6.7 mg/kg body weight Ketamine-Hydrochloride + Xylazine
- Animals fasted: No
- How many animals: all main group animals
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Leukocyte count, Thrombocyte count, Differential leukocyte count and red cell morphology, Reticulocyte count, Heinz bodies, Coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the study and after the recovery period
- Animals fasted: No
- How many animals: all recovery animals
- Parameters examined: Sodium, Potassium, Inorganic phosphorus, Uric acid, Bilirubin total, Bilirubin direct, Creatinine, Glucose, Urea, Calcium, Chloride, Aspartate aminotransferase (ASAT/GOT), Alanine aminotransferase (ALAT/GPT), Alkaline phosphatase (AP), Gamma-glutamyltranspeptidase (GGT), Cholesterol, Triglycerides, Total protein, Albumin

URINALYSIS: Yes
- Time schedule for collection of urine: overnight from day 25 to day 26 and day 39 to day 40
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance, Color, Volume, Specific weight, pH-Value, Hemoglobin, Protein, Glucose, Bilirubin, Urobilinogen, Ketone bodies, Sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly for general observations, end of study for FOBS
- Dose groups that were examined: all
- Battery of functions tested: sensory reactivity to stimuli of different types (auditory, visual, and proprioceptive), startle reflex (click response), response to approach with the finger to the nose of the animal, righting reflex, presence and absence of pupillary constriction, motor function including measurement of motor activity, and forelimb and hindlimb grip strength, motor activity during a 60-minute period in an 16-station automated motor activity monitoring 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.
The lungs were fixed endotracheally with formalin solution.

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

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

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)

Evaluation was performed by l/S Research, Hoechst Marion Roussel Deutschland GmbH, with the aid of a program package for the evaluation of toxicological studies.
The calculation methods used are referred to on the computer printouts.

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:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

only incidental findings (see below)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

discoloration of the urine due to excretion of the dyestuff

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

only incidental findings (see below)

Gross pathological findings:

no effects observed

Description (incidence and severity):

only incidental findings (see below)

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

only incidental findings (see below)

Histopathological findings: neoplastic:

no effects observed

Details on results:

Behavior, state of health and mortality
No deaths occurred throughout the study. Behavior and state of health remained unaffected by the administration of the test compound in all dose groups. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed.

Neurotoxicological examinations
Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, motor activity and forelimb and hindlimb grip strength were not influenced by the administration of the test compound in all groups.

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

Food and water consumption
Food and water consumption of all animals remained unaffected by the administration of the test compound throughout the study.

Hematological investigations
The hematological examination at the end of the treatment period did not show any compound-related effect.

Clinical chemistry
After 28 days of treatment, statistical evaluation of the serum parameters revealed increased values in creatinine in male animals of the high dose group, ALAT in all dosed males, and phosphorus in all dosed females. Decreases were observed in chloride of males and triglycerides of females of the high dose group.
At the end of the recovery period a statistically significant increase in calcium and chloride was found in high dose females.
All apparently statistically significant changes in serum parameter are within the range of the historical control data. Furthermore, the changes occurred in one sex only and were not found in the recovery group. Consequently, these findings have to be considered to be incidental and not substance related.

Urine analysis
Urine examination did not show any compound-related adverse effect; sediments were inconspicuous.
After 28 days of treatment with Diamantechtrot BT male animals of the high-dose group showed orange discolored urine.

Organ weights
Organ weights of the treatment groups were not adversely affected by administration of the test substance.
At the end of the recovery period the statistical evaluation of the absolute organ weight noted increased kidney weights in males of the high dose group.
The apparently higher absolute kidney weights in high-dose males is an incidental finding, not related to the treatment with Diamantechtrot BT. This is a consequence of the higher terminal body weight of this group. Furthermore, there were no corresponding changes observed after 28 days of treatment and this finding occurred in one sex only.

Macroscopic and microscopic findings
Single sporadic findings were greyish discolored lymph nodes in animal no. 56, and a cyst on the uterine cervix in animal no. 57 in the high dose group.
Histopathological sporadic findings were observed in various organs of different animals, which correlated to the gross pathology findings (pigment deposits in the lymph nodes), or could also be observed in animals of the control group; type and incidence of these changes were not relevant for the assessment of the test article.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects noted at any dose group

2

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Due to the orange discolored urine in male animals of the high-dose group after 28 days of treatment with Diamantechtrot BT

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Repeated administration of Diamantechtrot BT did not cause any compound-related adverse effects at any dose. With regard to the present study, the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg body weight per day.
Due to the orange discolored urine in male animals of the high-dose group after 28 days of treatment with Diamantechtrot BT the No Observed Effect Level (NOEL) is 250 mg/kg body weight per day.

Executive summary:

Observations and measurements

Groups of male and female rats received Diamantechtrot BT 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.

Behavior and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, water consumption once weekly.

Once before the first treatment and once a week thereafter 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.

Hematological 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 and recovery period each.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for

microscopically visible changes.

Body weights, hematological 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 analyzed with the aid of a statistical program to show differences compared to the controls.

Results

No deaths occurred throughout the study.

Behavior, state of health, body weight development, food and water consumption, organ weights, urinalysis as well as neurotoxicological, hematological and blood serum parameters remained unaffected by the administration of the test compound.

After 28 days of treatment with Diamantechtrot BT male animals of the high-dose group showed orange discolored urine.

Macroscopical and histopathological examination did not reveal any compoundrelated alteration.

Conclusion

In conclusion, repeated administration of Diamantechtrot BT did not cause any compound-related adverse effects at any dose.

With regard to the present study, the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg body weight per day.

Due to the orange discolored urine in male animals of the high-dose group after 28 days of treatment with Diamantechtrot BT the No Observed Effect Level (NOEL) is 250 mg/kg body weight per day.