REAKTIV-ORANGE DYPR 934

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:

1999-02-10 to 1999-03-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in compliance with EEC-Guideline B7 and OECD Guidelines for testing chemicals 407 according to the good laboratory practice regulations annex of paragraph 19a, section 1 of the chemical law of July, 25, 1994.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1999

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 (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Details on test material:

-Name of test material (as cited in study report): Reaktiv-Orange DYPR934

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: rat, Sprague-Dawley
-Strain: Sprague-Dawley Hsd:SD
-origin: Harlan Winkelmann, Gartenstr. 27 D-33178 Borchen (SPF breeding colony)
- Weight at study initiation: 426 g /mean
-n=15
- Water (e.g. ad libitum): tap water in plastic except for the period in which animals were kept in diuresis cages.
- Acclimation period: at least 7 days
-Food: Ssniff® Ms-H (V2233) ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): RT
- Humidity (%): 50 +/- 20 %
- Photoperiod (hrs dark / hrs light): 12 hours daily
Maintenance: in a fully air conditioned room in macrolon cages granulate in group 5 animals.
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.
-Randomization: randomization Scheme 98.0753
-Animal identification: fur marking with KMn04 and cage Numbering
-Stability and homogeneity in the vehicle: is guaranteed for 15 days according to report of Analytical Toxicology dated 1999-02-15 guidelines.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized water

Details on oral exposure:

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.
Preparation of the test compound
Dose Concentration Volume applied Vehicle
(mg/kg b.w.) In % (w/v) (mL/kg b.w.) Deionized water
0.0 0.00 5 Deionized water
62.5 1.25 5 Deionized water
250.0 5.00 5 Deionized water
1000.0 20.00 5 Deionized water
Reaktiv-Orange DYPR 934 was dissolved in the stated concentrations in deionized water at the following dates:
1) 1999-02-10
2) 1999-02-24
After each measurement of the body weight the calculation of the application volume was repeated.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

No. of animals per sex per dose:

DOSE LEVEL AND NUMBER OF ANIMALS:(M/F)

Group Dose (mg/kg b.w./d) Main groups Recovery groups
1 0.0 5 5 5 5
2 62.5 5 5 - -
3 250.0 5 5 - -
4 1000.0 5 5 5 5

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for road 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 934 in the rat yielded a medium lethal dose (LD50) above 2000 mg/kg body weight in both male and female animals

Examinations

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weights of the 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 (two 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 kg body weight over a 24 hour period.
WATER CONSUMPTION: water consumption was determined once weekly over a period of 16 hours (from approx. 3.15 p.m. to 7.15 a.m.)

WATER CONSUMPTION AND COMPOUND INTAKE Yes
- Time schedule for examinations:
HAEMATOLOGY: Yes
- Time schedule for collection of blood:
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 (intraperiteonal injection of B7 + 6.7 mg/kg body weight Ketamine-Hydrochloride + Xylazine). In order to prevent systemic errors, blood sampling was conducted in a randomized order.(see under Clinical chemistry)
The following hematological parameters were determined:
PARAMETER
ERYTHROCYTE COUNT
HEMOGLOBIN
HEMATOCRIT
MEAN CORPUSCULAR VOLUME (MCV)
MEAN CORPUSCULAR HEMOGLOBIN (MCH)
MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC)
LEUKOCYTE COUNT
THROMBOCYTE COUNT
DIFFERENTIAL LEUKOCYTE COUNT AND RED CELL MORPHOLOGY
REICULOCYTE COUNT
HEINZ BODIES
COAGULATION TIME



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. In order to prevent systematic errors, exsanguinations were conducted in a randomized order:
RANDOMIZATION OF CAGES FOR BLOOD SAMPLING AND DISSECTION
-RECOVERY VALUE
SEQUENCE CAGE
1 2
2 8
3 6
4 12

FINAL VALUE
SEQUENCE CAGE
1 11
2 4
3 10
4 9
5 7
6 1
7 3
8 5
- The following parameters were examined.
PARAMETER
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:
Urine analysis was performed on all animals a few days before termination of the study. For this purpose, the urine was collected by using metabolism cages (overnight from day 23 to 24). Food and water were withdrawn during this period.
Parameters checked in table [No 1] were examined:



NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: One 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 propioceptive) 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 papillary constriction was assessed using a pen flashlight directed into the light. Assessments of motor function were performed including measurement of motor activity, and forelimb and hindlimb grip strength. The animals were evaluated for 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.

Sacrifice and pathology:

NECROPSY AND MACROSCOPIC EXAMINATION:
After exsanguinations, 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.
HISTOPATHOLOGY
The following tissues or organs (or pieces of them) were preserved in a suitable fixative and processed for histopathological investigations:

HEART BRAIN URINARY BLADDER
LUNGS THYMUS LYMPH NODES ILIAC
LIVER TRACHEA LYMPH NODES (MANDIBULARE)
SPLEEN THYROID GLANDS WITH PARATHYROID GLANDS ADRENAL GLANDS
KIDNEYS TESTES PROSTATE GLAND
STOMACH EPIDIDYMIDES BONE MARROW (STERNUM)
JEJUNUM OVARIES N. ISCHIADICUS
COLON UTERUS SPINAT 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 I/S Research, Hoechst Marion Roussel Deutschland GmbH, with the aid of 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

Description (incidence and severity):

No death occurred throughout the study. Respiratory sounds were observed in some animals from the high dose group between day 11 and 19 of the study. Behavior and state of health remained unaffected by the administration of the test compound in the oth. g

Mortality:

no mortality observed

Description (incidence):

No death occurred throughout the study. Respiratory sounds were observed in some animals from the high dose group between day 11 and 19 of the study. Behavior and state of health remained unaffected by the administration of the test compound in the oth. g

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gains were slightly decreased in males from the high dose group. The changes were statistically significant between days 7 and 27 of the study. Body weight gains were comparable in the other groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable in all groups.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was markedly increased in both sexes from the high dose group during the treatment period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

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

Haematological findings:

no effects observed

Description (incidence and severity):

Statistical evaluation revealed decreases in hematocrit values in males of the intermediate and high dose group, decrease in MCV values in males from the high dose group, and increased reticuloyte values in males from the high dose group.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Increased ALAT values in males from the intermediate and high dose group and increased alkaline phosphate values in males from the high dose group. Decrease of CGT /decreases in albumin concentrations values (females from all dose group / female int. DG)

Urinalysis findings:

no effects observed

Description (incidence and severity):

Final value: High dose group (M/F) volume increased with a concomitant decrease in specific weight. pH-value tended to be higher. Recovery Value: decreased specific weights in males and increased in females. Same values as those of the control group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Neurotoxicological measurements including open fields observations, assessment of sensory function, motor activity, forelimb and hindlimb grip strength did not indicate any compound related effects.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Final value: decreased absolute kidney weights and increased relative brain weights (male/high dose group)-No clear dose dependency- A compound related effect is questionable.Recovery value: Spleen to body weight ratios were increased in males.

Gross pathological findings:

no effects observed

Details on results:

Clinical observations:
Behavior, state of health and mortality
No deaths occurred throughout the study. Respiratory sounds were observed in some animals from high dose group between day 11 and 19 of the study. Behavior and state of health remained unaffected by the administration of the test compound in other groups.
Opacity of the refracting media of the eyes and changes of the oral mucosa were not observed.

Body weight gain
Body weight gains were slightly decreased in males from the high dose group. The changes were statistically significant between days 7 and 27 of the study. Body weight gains were comparable in the other groups.

Neurotoxicological examinations:
Neurotoxicological measurements including open field observations, assessment sensory function, motor activity, forelimb and hind limb grip strength did not indicate any compound related effect.


Laboratory findings:
Clinical chemistry examinations revealed increases in ALAT activities in males from the intermediate and high dose group. Alkaline phosphates values were increased in male from the high dose group.
Urine volume was increased in both sexes from the high dose group, with a concomitant decrease in specific weight.
Organ weights were not obviously affected by administration of the test compound.
Four rats from the high dose group killed after the end of the treatment period showed an orange discolored stomach. Orange discolorations of the kidneys were observed in six recovery animals of high dose group.
Histopathological examination revealed dose-dependent sub mucosal mixed cellular infiltrations in the stomach of several animals from intermediate and high dose group killed after 28 days of treatment. Intratubular deposition of yellowish pigment in the kidneys was observed in several animals from the high dose group, particularly in the recovery group.

Organ Weights
Final value: Statistical evaluation revealed decreased absolute kidney weights and increased relative brain weights in males from the high dose group. These changes are considered to reflect the lower weight of these animals.
Absolute and relative adrenal weights were increased to a statistically significant degree in males from high dose group. However, there was no clear dose-dependency, and histopathological examinations did not reveal any compound-related effects. Therefore, a compound-related effect is questionable. The same applies to the statistically significantly increased absolute and relative liver weights in females of the intermediate and high dose group.
Recovery value: Spleen to body weight ratios were increased to a statistically significant degree in males.
Macroscopic and microscopic findings
Four rats from the high dose group killed after the end of the treatment period showed orange discolored stomach. Orange discolorations of the kidneys were observed in six recovery animals from the high dose group.
Histopathological examination revealed dose-dependent sub mucosal mixed-cellular infiltrations in the stomach of several animals from intermediate and high dose group killed after 28 days of treatment. Intratubular deposition of yellowish pigment in the kidneys was observed in several animals from the high dose group particularly in the recovery group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

62.5 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Repeated administration of Reaktiv-Orange DYPR 934 at the daily dose of 1000 mg/kg body weight caused slightly decreased body weights in males and markedly increased water consumption in both sexes.
Urine volume was increased, with a concomitant decrease in specific weight. Increased ALAT-and alkaline phosphate activities observed in male may indicate liver damage. However, histological examination of the liver did not reveal any compound-related change. Pigment deposition occurred in the kidneys. Furthermore, topical and reversible Irritation of the stomach was observed.
Slightly increased ALAT values were observed in males after repeated administration of Reaktiv-Orange DYPR 934 at the dose of 250 mg/kg body weight.
Slight irritation of the stomach was observed in both sexes from this dose group.
No compound-related effect was observed after repeated administration of Reaktiv-Orange DYPR 934 at the daily dose level of 62.5 mg/kg body weight.
Therefore the "No Observed Effect Level“(NOEL) is 62.5 mg/kg body weight.

Executive summary:

Groups of male and female Sprague-Dawley rats received Reaktiv-Orange DYPR 934 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.

The aim of this 29 -day toxicity study was to characterize the toxicological profile of Reactiv-Orange DYPR 934 after repeated oral exposure. Additionally, the results of this study can be used as a dose-range finding for subchronic and chronic toxicity studies.

During the study behavior 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 ("openfield"). Additionally the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth.

Furthermore neurotoxicological measurements, hematological and clinical examination and urine analysis were carried out 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.

Almost all the data were analyzed with the aid of a statistical program to show differences compared to the controls.

No deaths occurred throughout the study.

Neurotoxicological parameters were comparable in all groups.

Body weight gains were slightly lower in males from the high dose group. Food consumption was markedly increased in both sexes from the high dose group during the treatment period.

Hematological examinations did not reveal any abnormalities.

Histopathological examination revealed dose-dependent sub mucosal mixed cellular infiltrations in the stomach of several animals from intermediate and high dose group killed after 28 days of treatment. Intratubular deposition of yellowish pigment in the kidneys was observed in several animals from the high dose group, particularly in the recovery group.

In conclusion, repeated administration of Reaktiv-Orange DYPR 934 at the daily dose of 1000 mg/kg body weight caused slightly decreased body weights in males and markedly increased water consumption in both sexes. In addition, urine volume was increased, with a concomitant decrease in specific weight. Polyuria and polydipsia are well-known side effects for lithium. As the content of lithium is rather high in this test substance, these effects were most likely related to the lithium salt content.

Increased ALAT-and alkaline phosphate activities were observed in male only; however, histological examination of the liver did not reveal any compound-related change. Pigment deposition occurred in the kidneys; however, did not result in any organ damage.

Slight irritation of the stomach was observed in both sexes of the high-dose group.

No compound-related effect was observed after repeated administration of Reaktiv-Orange DYPR 934 at the daily dose level of 62.5 mg/kg body weight. No adverse substance-related effect was noted at a dose level of 250 mg/kg bw/day; hence, the "No Observed Effect Level“ (NOEL) is 62.5 mg/kg body weight and the "No Observed Adverse Effect Level“ (NOAEL) is 250 mg/kg body weight per day.

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