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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:
23 June 2004 to 06 October 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was diluted in corn oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Switzerland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: males 146 g (mean); females 127 g (mean)
- Fasting period before study: none
- Housing: In groups of five in Makrolon type-4 cages
- Diet (e.g. ad libitum): Pelleted standard rat maintenance diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


DETAILS OF FOOD AND WATER QUALITY: None of the contaminants analysed in the water and diet is considered to have been present at a concentration which would have affected the validity of the results.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70 %
- Air changes (per hour): 10-15 per hours
- Photoperiod (hours dark / hours light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. The test formulations were prepared weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on solubility study.
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): not specified
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 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.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 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 males and 5 females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based upon the results of a non-GLP 5-day dose- range-finding study (RCC Study Number 853872) in which CBI was administered by gavage to 2 rats per group and sex. Animals showed no clinical signs.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): random
Positive control:
not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and viability. General cage side observations once prior to administration then twice daily on days 1-3; once daily on days 4-28.
- Cage side observations checked included: mortality, viability and general clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first test item exposure and once weekly during weeks 1-3 in random order.

BODY WEIGHT: Yes
- Time schedule for examinations: at sacrifice

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: not specified
- Anaesthetic used for blood collection: Yes (isoflurane) / No / Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: not specified
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at week 4
- Dose groups that were examined: All rats
- Battery of functions tested: Hind-forelimb grip strength measurement was performed using a push-pull strain gauge. Locomotor activity was measured quantitatively. Decreased or increased activity was recorded. Activity was measured with an AMS Föhr Medical Instruments GmbH. Activity of the animals was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes were reported.


IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2)

HISTOPATHOLOGY: Yes (see table No 2)
Statistics:
The following statistical methods were used to analyse the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
-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.
-Fisher's exact-test was applied to the macroscopic findings.
The following statistical methods were used for statistical analysis of clinical laboratory data:
-Quantitative data were analysed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett.
Alternatively, if the variances are considered to be heterogenous (psO.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared
to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test
(psO.05).
-Ordinal data such as urine sediment were analysed using the KruskaI-Wallis test. If this test was significant (pcontrol group and each of the treatment groups using Dunn's test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test-item related clinical signs.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males at 1000 mg/kg/day body weight gain was decreased at weeks 2 and 3 of the treatment period. In females at 600 and at 1000 mg/kg/day, body weights were lower than those of the controls at week 2. Body weight gain was decreased at 600 and at 1000 mg/kg during weeks 2 and 3. These findings were considered to be test item-related, but were minor and of no toxicological relevance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related changes in food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in haematological data after 4 weeks that could definitively be related to treatment with the test item. All differences recorded were considered to be incidental and in general comparable with those of the controls and within limits of the historical reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was a slight to moderate reduction in total bilirubin levels in both sexes in all test item treated groups, a moderate increase in cholesterol levels and moderate to slight increase in phospholipid levels in females in all test item treated groups, a slightly lower GLDH activity in males at 600 and 1000 mg/kg/day and slightly lower K + and Ca++ level in females at 1000 mg/kg/day.

Slightly lower A/G ratios were noted in females at 1000 mg/kg/day, however, as there were no toxicologically relevant effects on the absolute levels of albumin or globulins, this was considered to be fortuitous and not related to treatment with the test item.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Grip Strength at week 4
In males at 600 mg/kg/day, the grip strength was reduced in the forelimb. In females, the grip strength was reduced in the forelimb at 200 mg/kg/day and in the hind limb at 600 and 1000 mg/kg/day. Since these findings were minor and there was no clear dose relationship, they were considered to be incidental and not related to treatment with test item.

Locomotor Activity
In females at 200 mg/kg/day, the locomotor activity was increased at 40 and 50 minutes. At 1000 mg/kg/day, the locomotor activity was increased only at 40 minutes.
Since these findings were minor and there was no clear dose relationship, they were considered to be incidental and not related to treatment with test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males at 200 and 600 mg/kg/day liver weights were increased. The liver/body weight ratio was increased at all dose levels, whereas the liver/brain weight ratio attained statistical significance only at 600 mg/kg. In females liver weights were increased absolutely and relatively at all dose levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic findings were evident at necropsy. All other macroscopic findings recorded were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the liver of males and females at 200, 600 and 1000 mg/kg/day, minimal to slight, mostly centrilobular hepatocellular hypertrophy was recorded. Increased incidence and/or severity (ranging from minimal to moderate degrees) of hepatic fatty change (periportal to diffuse) could be observed in male animals at 1000 mg/kg/day and females at 200, 600 and 1000 mg/kg/day. Increased incidence of minimal degree of bile duct proliferation was observed in males at 600 mg/kg/day and in females at 200 and 600 mg/kg/day. In the thyroid gland of most males at 1000 mg/kg/day (4/5) and single males at 200 mg/kg/day (215) and 600 mg/kg/day (1/5), minimal hypertrophic changes of the follicular epithelium could be observed. All other microscopic findings recorded were considered to be within the range of normal background lesions, which may be seen in rats of this strain and age. The observed effects were not considered to be adverse and of toxicological significance.
Histopathological findings: neoplastic:
not examined

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 adverse effect was observed.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the 28 -day repeated dose study, conducted according to an appropriate OECD Test Guideline and in compliance with GLP, a NOEL (no-observed-effect-level) could not be established for the test substance, however, no adverse effects were seen in this study and so the NOAEL (no-observed- adverse-effect-level) of the test substance was considered to be ≥ 1000 mg/kg.