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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
Study period:
18 JULY 2006 TO 8 SEPTEMBER 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13,1974, amended December 5,1986
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description Black sticky powder;
Purity Main component approx. 53%;
Storage conditions At room temperature (15 - 25 °C) in the original
container away from direct sunlight in an exsiccator.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test animal: Rat, HanRcc:WIST(SPF)
Total number of animals per group: Groups 1 and 4: 10 males; 10 females; groups 2 and 3: 5 males and 5 females
Total number of animals: 30 males and 30 females
Age at delivery: 6 weeks;
Body weight range (acclimatization): males: 130.9-158.6 g; females: 113.0-130.8 g
Conditions
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a target range for temperature of 22 ± 3 °C and for relative humidity between 30-70 %. There was 12 hours fluorescent light/ 12 hours dark cycle with music during the light period.
Accommodation: Groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding.
Diet: Pelleted standard Kliba no. 343 rat maintenance diet was available ad libitum (Batches no. 23/06 and 36/06).
Water: Community tap-water was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Oral, by gavage, 10ml/kg body weight, daily for 28 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Several application formulations were prepared at the test facility and representative analytical samples were collected and dispatched to the test site internally. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the
peak.
The identity of test item was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of test item in bidistilled water was demonstrated. The application formulations were considered to be stable for at least 2 hours and 7 days when kept under storage conditions.
In conclusion, the results obtained within this phase confirm the correct preparation and storage of application formulations during the conduct of this study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg body weight Group 2: 50 mg/kg body weight Group 3: 200 mg/kg body weight Group 4: 1000 mg/kg body weight
Basis:
nominal in water
No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females; groups 2 and 3: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
In this subacute toxicity study, test article was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. After termination of the treatment period half of the animals of the control group and of the high dose group were observed for a further 14-day treatment-free recovery period.
Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observation battery, locomotor activity and grip strength were performed during week 4.
At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and stomach and gross lesions from all animals.
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
Mortality/Viability: twice daily;
General cageside observation: once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).
Detailed clinical observations: in random sequence once before commencement of administration and once weekly (week 1-3) thereafter.
Food consumption: once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
Body weight: weekly during pretest, treatment and recovery and before necropsy.
Functional observational battery: during week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
Sacrifice and pathology:
All animals were weighted and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination.
Samples were collected from all animals at necropsy and fixed in neutral phosphate buffere 4% formaldehyde solution.
Other examinations:
Hematology, clinical biochemistry, urinalysis, absolute and relative organ weights, histotechnology and histopathology.
Statistics:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to the ophthalmoscopy data.
Student’s T-test was applied to grip strength and locomotor activity.

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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
Darkened urine color and increased urine pH were considered to be test item related changes. As both were reversible after the recovery period, they were considered to be not adverse.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences in the absolute or relative organ weights indicating an effect of test item after the treatment or recovery period.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Viability/Mortality: All animal survived until their scheduled necropsy.
General cageside observations: No clinical signs of adverse toxicological relevance were noted at any dose level.
Detailed clinical observation: No clinical signs of toxicological relevance were noted.
Functional observational battery: no test item-related adverse findings of toxicological relevance were noted.
Grip strength: no test item-related alterations in the mean fore-and hindlimb grip strength were recorded.
Locomotor activity: no test item-related effects in the mean locomotor activity were recorded.
Food consumption: no findings of toxicological relevance were noted.
Body weights: no test item-related changes of toxicological relevance were recorded.
Hematology: no test item-related effects in the hematological parameters were recorded.
Clinical biochemistry: no test item-related changes of toxicological relevance were noted.
Urinalysis: Darkened urine color and increased urine pH were considered to be test item related changes. As both were reversible after the recovery period, they were considered to be not adverse.
Organ weights: There were no differences in the absolute or relative organ weights.
Macroscopic findings: No macroscopic findings were considered to be test item-related.
Microscopic findings: No microscopic findings were considered to be test item-related.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

In this subacute toxicity study, the test article was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

After termination of the treatment period half of the animals of the control group and of the high dose group were observed for a further 14 -day treatment-free recovery period.

No treatment-related effects were noted on any parameter observed during the study (mortality, changes of toxicological relevance in detailed clinical observations, in grip strength and locomotor activity, in mean absolute and relative food consumption, in body weights and body weight gains, in hematology and clinical biochemistry parameters, in organ weights and ratios as well as in macroscopical and microscopical findings.

Test item-related findings were generally restricted to dark feces. Noted during general cage side observations and functional observational battery (week 4), in animals treated with 1000 mg/kg/day. In animals treated with 1000 mg/kg/day and in females treated with 200 mg/kg/day, the urine color was affected by the treatment, but was reversible after the recovery period. In animals treated with 1000 mg/kg/day, the urine pH was increased after the treatment, but was reversible after the recovery period.

Based on the results of this study, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of the test item was established as the no-observed-adverse-effect-level (NOAEL).