Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2010-03-29 to 2010-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

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:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Expiration date of the lot/batch: February 2011
- Storage conditions of test material: at room temperature, protected from light and under nitrogen gas in a dry and well-ventilated room.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder Janvier, Le Genest Saint-Isle, France
- Age at study initiation: on the day of treatment, the animals were approximately 8 weeks old
- Weight at study initiation: the animals had a mean body weight ± standard deviation of 213 ± 14 g
- Fasting period before study: 18 hours
- Housing: polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm)
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): drinking water filtered by a FG Millipore membrane
- Acclimation period: ar least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: 30 March 2010 To: 13 May 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 mg/mL and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: standard vehicle use for specific routes of administration
- Lot/batch no. (if required): MKBC 6753

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: In accordance with a Sponsor Representative requirement.

Doses:
300 and 2000 mg/kg.
No. of animals per sex per dose:
3 females per dose.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days after first day of dosing (D1)
- Frequency of observations and weighing: The animals were observed frequently during the hours following administration of the test item, for detection of possible treatment-related clinical signs. Thereafter, observation of the animals was made at least once a day.
Type, time of onset and duration of clinical signs were recorded for each animal individually.
The animals were weighed individually just before administration of the test item on day 1 and then on days 8 and 15.
- Necropsy of survivors and dead animals performed: yes

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
At 2000 mg/kg (first step), one female was found dead on day 6.
At 2000 mg/kg (second step), two females were found dead on day 3 or 4. The other female was sacrificed on day 8 for ethical reasons (severe clinical signs).
Clinical signs:
At 300 mg/kg (first and second step), no clinical signs were observed.

At 2000 mg/kg (first step), hypoactivity, piloerection, tremors, hypersensitivity to the touch, exophthalmia and rhinorrhea were noted in the female found dead on day 6. In the surviving animals, hypoactivity or sedation, piloerection, tremors, dyspnea, exophthalmia, rhinorrhea, ocular
secretion, hypersensitivity to the touch and ataxia were noted between day 3 and day 7 or 9.

At 2000 mg/kg (second step), piloerection, hypoactivity and dyspnea were noted in the female found dead on Day 4. No clinical signs were observed in the female found dead on Day 3. Piloerection, hypoactivity then sedation, dyspnea, cold to the touch and body weight loss (-26%) was observed in the third female.
Body weight:
When compared to CIT historical control data:
. a lower body weight gain (5 g vs. 15 ± 8 g in control data base) was noted between day 8 and day 15 in 1/6 female given 300 mg/kg,
. a lower body weight gain (8 g vs. 41 ± 9 g in control data base) was observed between day 1 and day 8 in 1/2 females treated at the dose-level of 2000 mg/kg. The body weight gain returned to normal thereafter.
. body weight loss was observed in 1/2 female treated at 2000 mg/kg.
Gross pathology:
Macroscopic examination of the main organs of the animals revealed no apparent abnormalities.

Applicant's summary and conclusion

Interpretation of results:
other: Toxicity category 4 / Harmful
Remarks:
Criteria used for interpretation of results: other: CLP (Reg. 1272/2008/EC) and Directive 67/548/EEC
Conclusions:
The oral LD50 of the test item was comprised between 300 and 2000 mg/kg in rats.
Executive summary:

The acute oral toxicity of the test item was evaluated in rats according to OECD (No. 423, 17th December 2001) and Commission Regulation (EC) (No. 440/2008, B.1tris, 30 May 2008) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.    

The test item was prepared in corn oil and was administered by oral route (gavage), under a volume of 10 mL/kg, to groups of three fasted female Sprague-Dawley rats. The study design was as follows:  The starting dose-level was 300 mg/kg bw. As no deaths occured, another assay was carried out on 3 animals at 2000 mg/kg bw . After this second assay as 1/3 animals died, the results were confirmed in 3 other animals at the dose-level of 2000 mg/kg bw. As all animals died in the third assay , another assay was carried out at 300 mg/kg bw.

At each step, clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration of the test item.   All animals were subjected to necropsy.

At dose-level of 300 mg/kg: first step (three females): No mortality and no clinical signs were noted at this dose-level. When compared to CIT historical control data, the body weight gain of the animals was not affected by treatment with the test item. At necropsy, no apparent abnormalities were observed in any animal.

At dose-level of 2000 mg/kg: first step (three females): One female was found dead on day 6. Hypoactivity, piloerection, tremors, hypersensitivity to the touch, exophthalmia and rhinorrhea were noted prior to its death. In the surviving animals, hypoactivity or sedation, piloerection, tremors, dyspnea, exophthalmia, rhinorrhea, ocular secretion, hypersensitivity to the touch and ataxia were noted between day 3 and day 7 or 9. A body weight loss (-8%) was noted in 1/2 surviving animals between day 1 and day 8. When compared to CIT historical control animals, a lower body weight gain (vs. 41 ±in control data base) was observed in the other female between day 1 and day 8. In both animals, the body weight gain returned to normal between day 8 and day 15. At necropsy, no apparent abnormalities were observed in any animal.
In the s
econd step (confirmation on three other females
): Two females were found dead (day 3; day 4). Piloerection, hypoactivity and dyspnea were noted prior to the death (day 4) of only one of them. No clinical signs were observed before death of the other female. Piloerection, hypoactivity then sedation, dyspnea, cold to the touch and body weight loss (-26%) was observed in the third female. According to the severe clinical signs observed, this animal was sacrificed on day 8 for ethical reasons. At necropsy, no apparent abnormalities were observed in any animal.  

At dose-level of 300 mg/kg: second step (confirmation on three other females): No mortality and no clinical signs were observed at this dose-level. When compared to CIT historical control data, a lower body weight gain (vs. 15 ±in control data base) was noted in 1/3 females between day 8 and day 15. At necropsy, no apparent abnormalities were observed in any animal.  

Under these experimental conditions, the oral LD50of the test item was comprised between 300 and 2000 mg/kg in rats.