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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:
12-April-2006 to 22-August-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:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to guideline
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
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: Black powder
Stability of test item: Hygroscopic; stable under storage conditions
Storage conditions: at room temperature (15 - 25°C) in a desiccator in the original container away from direct sunlight.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system: Rat, HanRcc:WIST (SPF)
Total number of animals/group: Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females
Total number of animals used: 30 males and 30 females
Age at delivery: 6 weeks
Body weight range at acclimatization: Males: 130.4 - 159.2 grams (mean 145.7 grams)
Females: 115.9 - 130.4 grams (mean 121.4 grams)
Identification: Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo
Randomization: Computer-generated random algorithm
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry
Conditions: Standard Laboratory Conditions. Air-conditioned with 10 - 15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ±3 °C; relative humidity range: 30 - 70 %). There was 12-hour fluorescent light/12 -hour dark cycle with music during the light period.
Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
Water: Community tap-water from Itingen was available ad libitum in water bottles.
Diet: Pelleted standard Provimi Kliba 3433 (batch no.01/06) rat maintenance diet was available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method: Oral, by gavage.
Daily dose levels: 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
Frequency of administration: Daily
Dose volume: 10 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 3 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200 and 1000 mg/kg body weight
Basis:

No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only.
The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Mortality / Viability
Observations for mortality/viability were recorded twice daily.
General Cageside Observations (Daily)
The animals were observed for clinical signs 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 (Weekly)
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1 - 3) thereafter.
Food Consumption
The food consumption was recorded 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 Weights
Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.
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 anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory investigations, organ weights and ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were 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) were applied instead of the Dunnetttest when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings.

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
Behaviour (functional findings):
not specified
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 specified
Details on results:
MORTALITY / VIABILITY
All animals survived until scheduled necropsy.
CLINICAL SIGNS
During daily observations, dark feces (with dose-related time of onset and severity) were noted in males and females treated with 200 mg/kg/day or 1000 mg/kg/day. In the latter group, this finding was observed from days 1 - 5 of recovery with diminishing severity. Dark feces were considered to be a passive test item-related effect without toxicological relevance.
During weekly observations (weeks 1-3), no signs were noted at any dose level.
FUNCTIONAL OBSERVATIONAL BATTERY
During the functional observational battery (week 4), no signs were noted at any dose level.
Grip Strength
No test item-related changes in the mean fore- or hindlimb grip strength were noted at any dose level.
Locomotor Activity
Minor differences noted in the mean locomotor activity of the test item-treated rats were considered to be of no toxicological relevance.
FOOD CONSUMPTION
No differences of toxicological relevance were noted in the mean absolute or relative daily food consumption at any dose level tested.
BODY WEIGHT
The mean body weights of the test item-treated rats compared favorably with those of the control rats.
CLINICAL LABORATORY INVESTIGATIONS
Hematology
No test item-related changes were noted in the hematology parameters at any dosen level.
Clinical Biochemistry
At 1000 mg/kg/day, reductions in blood glucose, increased total bilirubin, elevated cholesterol, triglycerides (females only), phospholipids (males only) and phosphorus were noted after four weeks' treatment. With the exception of total bilirubin, all differences remained within the ranges of the historical control values, but may indicate minor adaptive changes in liver metabolism. The elevation in total bilirubin was considered to be a possible artifact induced by staining of the plasma.
Urinalysis
No test item-related changes were noted in the urinalysis parameters at any dose level.
ORGAN WEIGHTS
No differences of toxicological relevance were noted in the mean absolute or relative organ weights at any dose level after four weeks' treatment or two weeks' subsequent recovery.
MACROSCOPIC / MICROSCOPIC FINDINGS
At the end of the treatment period, a violet discoloration of the ileum, cecum, colon and rectum was recorded in all males and females treated with 1000 mg/kg/day, while black discoloration of the kidneys was seen in all males and females treated with 1000 mg/kg/day at the end of treatment and recovery periods. Since the microscopic examination of intestinal organs did not reveal any changes associated to the discoloration, no toxicological meaning was attributed to it.
Microscopically patchy fatty change (with small droplets) was seen in the liver of animals treated with 1000 mg/kg/day. After two weeks of recovery, the liver of males returned to normal, whereas changes were still present in a few females. Eosinophilic granules (hyaline droplets) were observed in the renal tubules (mainly proximal) of some male rats treated with 1000 mg/kg/day. In addition dark pigments were seen in the renal tubules of some males and females treated with 1000 mg/kg/day. Neither granules nor pigmentation were associated with any other morphological change such as degeneration or inflammation. The pigmentation was probably associated to the black discoloration reported at necropsy. After two weeks of recovery, the kidneys returned to normal.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Dark feces observed at 200 mg/kg bw and above were considered to be a passive test item-related effect without toxicological relevance.
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 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 as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, treatment and recovery periods. Functional observational 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 all gross lesions from all animals.

From the animals of the low and middle dose groups, liver and kidneys were examined to establish a no-effect level.

And the oral administration of the test item to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in no mortality, no clinical signs of toxicological relevance during daily or weekly observations (weeks 1-3), no effects upon functional observational battery (week 4), including changes in grip strength and locomotor activity. The mean daily food consumption and mean body weights were unaffected. The organ weights and ratios of test item-treated rats compared favorably to those of the controls.

No test item-related changes were noted in the hematology parameters at any dose level. All differences of clinical biochemistry remained within the ranges of the historical control values, but may indicate minor adaptive changes in liver metabolism. The elevation in total bilirubin was considered to be a possible artifact induced by staining of the plasma. No test item-related changes were noted in the urinalysis parameters at any dose level. No differences of toxicological relevance were noted in the mean absolute or relative organ weights at any dose level after four weeks' treatment or two weeks' subsequent recovery. No test item-related changes were noted in the macroscopic/microscopic findings.

Based on the results of this study, 50 mg body weight/dayof the test item was established as the no-observed-effect-level (NOEL), and1000 mg/kg body weight/day of the test item as the no-observed-adverse-effect-level (NOAEL).

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