Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 130.9 -161.4 grams (mean 154.0 grams); Females: 112.2 -128.5 grams (mean 120.1 grams)
- Fasting period before study: not specified
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet: Pelleted standard Provimi Kliba 3433 (batch no. 84/02) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared daily.The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added (weight/volume). The mixtures were prepared using a magnetic stirrer and used at room temperature (17-23°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dose volume: 5 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200 and 1000 mg/kg bodyweight
Basis:
actual ingested
No. of animals per sex per dose:
10: control and 1000 mg/kg bodyweight
5: 50 and 200 mg/kg bodyweight
Control animals:
yes, concurrent vehicle
Details on study design:
The groups comprising 5 animals per sex 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:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality/viability were recorded twice 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: Yes
- Time schedule: Performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during pretest, treatment and recovery and before necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain: Yes

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks and after 6 weeks (recovery group)
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 hours

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks and after 6 weeks (recovery group)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 hours

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all
- Battery of functions tested: grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, 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 Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test were applied to the macroscopic findings.

The following statistical methods were used for statistical analysis of clinical laboratory data:
• Quantitative data were analyzed 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 (p<0.05), anon-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 (p<0.05).

Results and discussion

Results of examinations

Details on results:
MORTALITY / VIABILITY
All animals survived until scheduled necropsy.

CLINICAL SIGNS
No test item-related findings were noted during daily and weekly observations (weeks 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY
Grip Strength: The fore- and hindlimb grip strength values of all rats compared favorably.
Locomotor Activity: No test item-related changes of toxicological relevance were noted at any dose level tested.

FOOD CONSUMPTION
The mean daily food consumption of all rats compared favorably.

BODY WEIGHT
Slightly lower mean body weights and lower body weight gain was noted in males treated at 200 mg/kg/day and 1000 mg/kg/day when compared with controls. These changes were considered to be test item related. The mean body weight gain during the recovery period was similar in all groups.

CLINICAL LABORATORY INVESTIGATIONS
Hematology:
No test item-related changes in the hematology parameters were noted at any dose level tested.
Clinical Biochemistry:
Increased gamma glutamyl transferase activity was noted after four weeks' treatment in females at 200 mg/kg/day and in both sexes at 1000 mg/kg/day when compared with the controls. This finding was considered to be a test item-related adaptive change, and was reversible after two weeks'recovery. All other clinical biochemistry parameters were unaffected.
Urinalysis:
None of the minor differences in the urine parameters were considered to be of toxicological relevance.

ORGAN WEIGHTS
Elevated absolute liver weights were noted in females treated with 200 mg/kg/day, and in both sexes treated with 1000 mg/kg/day. Liver-to-body weight ratios were increased in females treated with 50 mg/kg/day, 200 mg/kg/day and in both sexes treated with 1000 mg/kg/day, whereas elevated liver-to-brain weight ratios were noted in females treated with 200 mg/kg/day, and in both sexes treated with 1000 mg/kg/day. These changes were reversible and were therefore considered to be indicative of metabolic adaptation and of no toxicological relevance. Elevated absolute and relative spleen weights were noted in females treated with 200 mg/kg/day and 1000 mg/kg/day. These changes were, in the absence of microscopical alterations, considered to be of no toxicological relevance. All other minor differences in organ weights were considered to be either adaptive changes or fortuitous.

MACROSCOPIC / MICROSCOPIC FINDINGS
At the end of the treatment and following recovery period, no test item-related gross lesions were observed. Under the conditions of this study, the test substance induced histopathological changes in liver, thyroid gland, and stomach. In the liver, an increased incidence of a minimal to slight hepatocellular hypertrophy was recorded in animals treated with 1000 mg/kg/day, which correlated to increased organ weight. After the recovery period, this change reverted to normal levels. It was regarded as an adaptive change most likely caused by the metabolic biotransformation of the test item. In the thyroid gland, a minimal to slight hypertrophy of the follicular epithelium was recorded in males treated with 1000 mg/kg/day. It was regarded as an adaptive change most likely caused by compensation of an increased degradation of thyroid hormones. After the recovery period, the incidence of follicular hypertrophy was largely reduced. with 1000 mg/kg/day. The slightly dilated crypts often were filled with mucus. After the recovery period, the glandular ectasia was not recorded, therefore this finding was regarded as an adaptive effect. None of these findings were considered to be of adverse character.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of the test substance to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in slightly lower body weight development in males treated with 200 or 1000 mg/kg/day for four weeks. In addition, adaptive changes in clinical chemistry and liver weights were observed. Based on the results the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

The test substance 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, corn oil, 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. No treatment-related clinical signs or gross necropsy findings were observed for either sex at any dose level. No alterations related to treatment in other measurements including grip strength, locomotor activity, food consumption, hematology or urine analysis were observed in any dose group for either sex.

Slightly lower mean body weights and lower body weight gain was noted in males treated at 200 mg/kg/day and 1000 mg/kg/day when compared with controls. The mean body weight gain during the recovery period was similar in all groups. Changes in clinical chemistry (increased gamma glutamyl transferase activity in females at 200 mg/kg/day and in both sexes at 1000 mg/kg/day) and organ weights (elevated liver-to-body weight ratios in females treated with 50 mg/kg/day, 200 mg/kg/day and in both sexes treated with 1000 mg/kg/day) were considered to be adaptive and were reversible after two weeks' recovery. The elevated liver weights correlated with an reversible increased incidence of a minimal to slight hepatocellular hypertrophy in animals treated with 1000 mg/kg/day. This finding was also considered to be adaptive in nature. Elevated absolute and relative spleen weights were noted in females treated with 200 mg/kg/day and 1000 mg/kg/day. These changes were, in the absence of microscopical alterations, considered to be of no toxicological relevance. In the thyroid gland, a minimal to slight hypertrophy of the follicular epithelium was recorded in males treated with 1000 mg/kg/day. It was regarded as an adaptive change most likely caused by compensation of an increased degradation of thyroid hormones. After the recovery period, the incidence of follicular hypertrophy was largely reduced. In the stomach, a minimal to slight glandular ectasia was recorded in some males treated with 1000 mg/kg/day. The slightly dilated crypts often were filled with mucus. After the recovery period, the glandular ectasia was not recorded, therefore this finding was regarded as an adaptive effect. None of these findings were considered to be of adverse character.

In conclusion, oral administration of the test substance to Wistar rats at doses of 200 and 1000 mg/kg/day resulted in non-adverse adaptive changes in liver, stomach, thyroid and in clinical chemistry parameters. Based on this results, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg/day.