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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:
13 October 2009 - ...........................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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
Remarks:
deviations to study plan but not to guideline
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Remarks:
idem above
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: brown black liquid
- Lot/batch No.: 01/07/09
- Expiration date of the lot/batch: 01/07/2011
- Storage conditions of test material: at room temperature
- Purity/Impurities: not applicable (complex composition)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: males had a mean body weight of 240 g (range: 230 g to 249 g) and females had a mean body weight of 197 g
(range: 180 g to 211 g).
- Fasting period before study: no
- Housing: individual suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France),
was placed under each cage. The cages were placed in numerical order on the racks.

- Diet (e.g. ad libitum): access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 6 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): 12 cycles per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: 22 October 2009 To: 24 February 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- the container was heated at 80°C for 65 minutes in a water bath and under magnetic stirring until complete dissolution of particles,
- the absence of crystallized particles was checked,
- as no crystallized particles persisted, the temperature of the water bath was not increased,
- after complete dissolution of crystallized particles, the appropriate amount of test item was taken in brown flasks and stored at room temperature
until use.

The test item was administered as a solution in the vehicle. Before use (weighing, sampling or dilution for the study), each sample of test item was
heated and stirred at 80°C under the same experimental conditions until obtaining a solution.

Fresh dosage form preparations were made extemporaneously of each administration, then the dosage forms were stored at room temperature, and delivered to the study room in brown flasks.

VEHICLE
- Concentrations in vehicle: 12.5, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- nature of analytical method: Ionic Chromatography, validated
- test item concentration: in weeks 1 and 4, within an acceptable range of [-10.2% to -6.0%] of variation compared to the nominal values
- homogeneity: not assessed, dosage form is a solution
- stability: not assessed, dosage form prepared extemporaneously each day of treatment.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 250 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were selected in agreement with the Sponsor on the basis of the results of a 14 day toxicity preliminary
study by oral route in rats (CIT/Study No. 36054 TSR). The test item was administered to three groups of three male and three female Sprague-Dawley rats at the dose level of 62.5, 250 or 1000 mg/kg/day under a dose-volume of 5 mL/kg. A control group was treated with the vehicle under the same experimental conditions.
No unexpected mortality occurred during the study in any animals. No clinical signs were observed. Body weight and food consumption were not affected by the treatment with test item. There were no treatment-related organ weights and macroscopic changes in any animals.

- Rationale for animal assignment (if not random): computerized stratification procedure.
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule:
mortality/morbidity: once a day during the acclimation period and at least twice a day during the treatment period
clinical signs : once a day

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
Body weight was recorded once before group allocation, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION: Yes
Food consumption was recorded once a week during the study.

HAEMATOLOGY, blood biochemistry and urinalysis: Yes
Hematology, blood biochemistry and urinalysis investigations were performed on all animals at the end of the treatment period.

NEUROBEHAVIOURAL EXAMINATION: Yes
A Functional Observation Battery (FOB), including detailed clinical examination, assessment of reactivity to manipulation and different stimuli and motor activity, was evaluated in each animal once at the end of the treatment period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table (see below) for animals of the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period

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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
non-adverse
Histopathological findings: neoplastic:
not examined
Details on results:
HISTOPATHOLOGY: NON-NEOPLASTIC
At the high-dose, microscopic findings related to local effect of test item were observed in the forestomach and consisted of minim epithelial acanthosis in most males and one female.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed at highest tested dose.
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: At the high-dose, microscopic findings related to local effect of test item were observed in the forestomach and consisted of minim epithelial acanthosis in most males and one female. Non adverse.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral route for 29 days to Sprague-Dawley rats at the dose levels of
62.5, 250 and 1000 mg/kg/day under a dosage-volume of 5 mL/kg/day.

No relevant in vivo findings were noted in any of the test item-treated animals. Hematology, blood biochemistry and urinalysis parameters were not
considered to be affected by the treatment with the test item. There were no treatment-related organ weights and macroscopic changes.

At the high-dose, microscopic findings related to local effect of test item were observed in the forestomach and consisted of minim epithelial
acanthosis in most males and one female.

At the intermediate- and low-dose, no treatment-related microscopic changes were observed.

The No Observable Effect Level (NOEL) was established at 250 mg/kg/day and the No Adverse Observable Effect Level (NOAEL) was established at
1000 mg/kg/day.
Executive summary:

The potential toxicity of the test item following daily oral administration (gavage) to rats for 4 weeks, was evaluated according to OECD (No. 407, 03 October 2008) and Commission Regulation (EC) (No. 440/2008, B.7, 30 May 2008) guidelines.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

The test item was administered daily by oral route for 29 days to 3 groups of

five male and five female Sprague-Dawley rats at the dose-levels of 62.5, 250 and 1000 mg/kg/day under a dosage‑volume of

5 mL/kg/day. Another group of five males and five females received the vehicle, purified water, under the same experimental conditions and acted as control group.

The test item was administered as a solution in the vehicle and the dosage form preparations were made extemporaneously before each administration. Each control and test item dosage form preparation was analyzed for concentration of the test item in weeks 1 and 4.

Animals were checked at least twice a day for mortality and at least once a day for clinical signs. A detailed clinical examination was performed once a week.A Functional Observation Battery (FOB), includingdetailed clinical examination, assessment of reactivity to manipulation and different stimuliand motor activity,was evaluated in each animal once at the end of the treatment period.

Body weight was recordedonce before group allocation, on the first day of treatment, and then once a week until the end of the study. Food consumption was recorded once a week during the study. Hematology, blood biochemistry and urinalysis investigations were performed on all animals at the end of the treatment period. On completion of the treatment period, the animals were sacrificed and a complete macroscopicpost‑mortemexamination was performed on all animals. Selected organs were weighed and designated tissues specimens were preserved. A microscopic examination was performed on designated tissues from control and high-dose animals and on all macroscopic lesions and on stomach with forestomach from low- and intermediate-dose animals.

The test item concentrations in administered dosage forms remained within an acceptable range of variation when compared to the nominal values. No unscheduled mortality occurred during the study. No relevant clinical signs related to treatment with the test item were noted.There were no signs of disturbance of neurobehavior in any of the test item-treated animals. Body weight and food consumption were not affected by the treatment with the test item. Hematology, blood biochemistry and urinalysis parameters did not reveal any toxicologically significant effect of the test item. No important or statistically significant changes in organ weights or organ-to-body weight ratios and no treatment-related necropsy findings were noted in any of the treated animals. A minimal acanthosis was noted in the limiting ridge of the forestomach, extending into the forestomach, in 4/5 males and 1/5 females treated at 1000 mg/kg/day with occasional increase in exocytosis of small mononuclear cells. No such change was noted inforestomachof animals treated at 62.5 or 250 mg/kg/day. This finding may be related to a local effect of the test item. Due to the very limited magnitude of this change, it was not considered to be adverse to the health of the affected animals.

The test item, was administered daily by oral route for 29 days to Sprague-Dawley rats at the dose‑levels of 62.5, 250 and 1000 mg/kg/day under a dosage-volume of 5 mL/kg/day.  

No relevantin vivofindings were notedin any of the test item-treated animals. Hematology, blood biochemistry and urinalysis parameters were not considered to be affected by the treatment with the test item.There were no treatment-related organ weights and macroscopic changes. At the high-dose, microscopic findings related to local effect of test item were observed in the forestomach and consisted of

minim epithelial acanthosis in most males and one female. At the intermediate- and low-dose, no treatment-related microscopic changes were observed. The No Observable Effect Level (NOEL) was established at 250 mg/kg/day and the No Adverse Observable Effect Level (NOAEL) was established at 1000 mg/kg/day.