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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
September 19, 1984
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid
- Analytical purity: commercial grade
- Lot/batch No.: Op. 11006
- Expiration date of the lot/batch: December 31, 1995
- Stability under test conditions: stable for at least 2 hours
- Storage condition of test material: room temperature, dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: KFM Kleintierfarm Madoerin AG, Fuellinsdorf/Switzerland
- Age at study initiation: 7-9 meeks
- Weight at study initiation: males: 147 - 173 g, females: 157 - 181 g
- Fasting period before study:
- Housing: Individually
- Diet (e.g. ad libitum): Pelleted standard Kliba no. 343 Batch 44/86 rat maintenance diet ('Kliba', Klingentalmuehle AG, Kaiseraugst, Switzerland), at
libitum.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, polyethylene glycole (PEG 400), was added. The mixture was prepared daily prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
Concentrations, homogeneity, and stability were checked by RCC analytical laboratories for all preparations.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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
Details on study design:
- Dose selection rationale:
Based upon data received from the following acute studies, carried out at RCC:
- acute oral toxicity in rats, RCC Project 062245, Ciba-Geigy Project 85 1241
- acute dermal toxicity in rats, RCC Project 062278, Ciba-Geigy Project 85 1244
In addition, data provided by the sponsor showed, that a test article (TK 12970/B) which is similar to the test compound had a "no observable effect level" of 100 mg/kg body weight in a 28-day oral toxicity study (GU-Project. No. 84 1066).
- Section schedule rationale (if not random): Computer-generated random algorithm

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality were recorded twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs of toxicity were assessed twice daily. A description of any abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded weekly during the acclimation and treatment period using an on-line
electronic recording system.

FOOD CONSUMPTION: Yes
- The food consumption was recorded once during the acclimation period and weekly thereafter using an online electronic recording system.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed at termination of treatment.
Ten minutes after the application of a mydriatic solution (Dispersa AG, Winterthur / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, Allschwil / Switzerland)
- Dose groups that were examined: Ophthalmoscopic examinations were performed on all animals.

HAEMATOLOGY: Yes, from the retro-orbital plexus
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes, under light ether anesthesia
- Animals fasted: Yes, the animals were fasted for 18 hours before blood sampling but water was provided.
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes, the animals were fasted for 18 hours before blood sampling but water was provided.
- How many animals: all animals
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks, over an 18-hour period into a specimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals were necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed
by experienced prosectors supervised by a pathologist. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination after 4 weeks.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution.
Adrenal glands, Heart, Kidneys, Liver, Spleen, Gross lesions.
Where there was a compound related alteration in an organ, this organ was histologically examined in all animals of all dose groups.

Organ weights: The following organ weights were taken from all animals necropsied at termination of treatment: Adrenal glands, Kidneys,
Liver, Testes.

HISTOPATHOLOGY: Yes
Slides of all tissues collected at terminal sacrifice from the animals of the control and high-dose groups as well as all animals which died spontaneously were examined by a pathologist. Treatment-related morphologic changes in the organs of any highdose animal required histological evaluation of the same organs in lower dose groups until a no-effect-level was determined. All abnormalities were described and included in the report.

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the pathologist.
Other examinations:
No other examinations were performed.
Statistics:
The following statistical methods were used to analyze the body weights, food consumption and organ weights:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Starting with day 21 until termination of treatment all rats of the high dose group (1000 mg/kg bw) showed red discolored extremities. In addition red
discolored feces were observed in the same animals betmeen day 18 and termination of the test. No other symptoms related to test article treatment were observed.

Treatment-unrelated signs and symptoms:
Prior to death, one female animal of the group with 300 mg/kg showed moderate sedation (days 17-19), rales (days 18-19) and moderate ruffled fur (days 17-19).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Due to a possible intubation error, two female animals, one of the control group and one of the group 300 mg/kg died spontaneously at day 17 respectively 20 of test.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The assessment of biochemical data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the clinical biochemistryparameters were considered to be incidental and of normal biological variation.
Description (incidence and severity):
The assessment of urinalysis data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
HISTOPATHOLOGY:
- No treatment related microscopic findings were recorded. From the tissues examined no cause of death could be established for the two rats which died. The various spontaneous microscopic findings recorded are within the normal range observed in this age and strain of rat. They can be attributed to subclinical infections, spontaneous congenital abnormalities or physiological status.
The nephroblastoma which mas recorded in one male of the 300 mg/kg body weight group is a seldom encountered malignant kidney tumor which occurs spontaneously with a Iow incidence in many rat strains and is not related to treatment.

Effect levels

Key result
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: No effects were observed at the limit dose.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based upon the results obtained in the study, the NOAEL for the 28-day repeated dose oral toxicity test of the substance was considered to be higher than 1000 mg/kg/day for male and female rats when administered orally by gavage.
Executive summary:

In a study according to OECD Test Guideline 407 under GLP conditions, Wistar rats were treated with three doses of the substance (100, 300 and 1000 mg/kg bw). The dose were applied by oral (gavage) daily for four weeks. Besides some clinical findings caused by the colour of the substance, no treatment-related effects were observed. Therefore, the NOAEL was considered to be higher than 1000 mg/kg/day for male and female rats.