Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 2014 - 10 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rat, 10 weeks old
- Weight at study initiation: 206 -224 g
- Fasting period before study: day before treatment
- Housing: Group caging (3 animals/cage)
- Diet (ad libitum): Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany.
- Water (ad libitum): tap water from municipal supply, as for human consumption from bottle ad libitum.
- Acclimation period: 19 days in first step, 20 days in second step and 21 days in third step

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15 (by central air-condition system)
- Photoperiod: Artificial light, from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
All doses were formulated in the vehicle. Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw. The test item was applied in a concentration of 500 mg/mL and 200 mg/mL. Formulations were prepared just before the administration.
Doses:
Starting dose was selected on the basis of the available information about the test item (Preliminary study: LD50: 2000-5000 mg/kg bw, based on sponsor’s information). The starting dose was 5000 mg/kg bw. One animal was treated with 5000 mg/kg bw dose. The first animal died, so dosing was proceeded at 2000 mg/kg bw in accordance with the flow charts in Annex 2 of the OECD guideline No. 423. Three animals were treated with 2000 mg/kg bw dose. Only one animal died in the second step at 2000 mg/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. Only one animal died in the third step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) was met.
No. of animals per sex per dose:
5000 mg/kg bw: 1 female
2000 mg/kg bw: 2 test groups 3 females each
Control animals:
no
Details on study design:
Mortality
Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and twice each day for 14 days thereafter.

General state, external appearance, behavior and clinical symptoms
Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h, after the treatment and once each day for 14 days thereafter. Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weight
The body weights were recorded on day 0 (just before the treatment), on day 1, on day 2, on day 7 and on day 15 with a precision of 1 g.

Necropsy
At the end of the observation period rats were sacrificed under isofluran anaesthesia. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size.
Statistics:
Evaluation
The method used is not intended to allow the calculation of a precise LD50 value.
The test item was ranked into classes of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423.
The frequency of the clinical symptoms was summarized in tabular form. The mean of the body weight and body weight gain were calculated by Excel spreadsheet software. Necropsy findings were described and summarised in tabular form.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 2 000 - <= 5 000 mg/kg bw
Mortality:
One rat (No.: 491) dosed at 5000 mg/kg bw (step 1) died on Day 0, 1 hour after the treatment.
Two out of six rats dosed 2000 mg/kg bw died. Animal No.: 498 of group 1 died on Day 2 and animal No.: 510 died on Day 1.
All deaths seemed to be consequences of systemic toxic effect of the test item.
Four rats (No.: 497, 506, 493, 511) survived until the end of the 14-day observation period.
Clinical signs:
In group 1 treated with 5000 mg/kg bw dose clinical signs of reaction comprised of decreased activity (2 cases of 2 observations), pain reaction (2/2), tonic convulsion (1/2), clonic convulsion (1/2), abnormal gait (2/2), incoordination (1/2), prone position (2/2), decreased grip- and limb tone (2/2), decreased body tone (2/2), decreased abdominal tone (2/2), decreased skin turgor (2/2), closed eyes (2/2), pale skin and mucous membrane (2/2), dyspnoea (2/2) and piloerection (2/2). These symptoms were observed on the treatment day between 30 min. and 1 hour after the treatment.

In group 2 treated with 2000 mg/kg bw dose clinical signs of reaction comprised of abnormal gait (19 cases of 44 observations), crouching (19/44), closed eyes (3/44), piloerection (19/44), decreased activity (8/44), incoordination (4/44), decrease body tone (3/44), decreased abdominal tone (3/44), pale skin and mucous membrane (5/44) and dyspnoea (5/44). These symptoms were observed between the treatment day and Day 1.

In group 3 treated with 2000 mg/kg bw dose clinical signs of reaction comprised of abnormal gait (17 cases of 43 observations), decreased activity (14/43), incoordination (12/43), crouching (17/43), decreased grip- and limb tone (6/43), decrease body tone (7/43), decreased abdominal tone (7/43), closed eyes (14/43), pale skin and mucous membrane (13/43) piloerection (17/43), and dyspnoea (3/43). These symptoms were observed between the treatment day and Day 1.
Body weight:
The body weight and body weight gain data of group 1 (5000 mg/kg bw) could not be evaluated, because of mortality.
In group 2 and 3 (2000 mg/kg bw) the mean body weight of the survivor animals corresponded to their species and age throughout the study.
Gross pathology:
The animal (No.: 491) treated with 5000 mg/kg bw dose and two animals (No.: 498, 510) treated with 2000 mg/kg bw dose died spontaneously during the study. Four animals (No.: 497, 506, 493, 511) treated with 2000 mg/kg bw dose were sacrificed scheduled during the study.
External (piloerection, pale skin and mucous membrane) and internal (pale lung, pale liver, pale spleen, pale kidneys) necropsy findings was observed in animal No.: 491 of group 1.
External (piloerection, blood around the nose) and an internal (autolysis) necropsy findings was observed in two animals (No.: 498, 510) of group 2 and group 3, respectively. Autolysis is normal physiological process after death.
Slight hydrometra was observed in female No.: 497 of the group 2. It is physiological finding and connected to the cycle of the animal.
Internal necropsy finding as pale kidneys was observed in two survivor animals (No.: 497, 511) of group 1 and group 2, respectively. This alteration could not be related to the test item toxic effect, but was regarded an individual variation. Most likely the observation is a congenital anomaly.
No pathological changes were found related to the effect of the test item during the macroscopic examination of survivor animals
Other findings:
No other findings

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
The acute oral LD50 value of the test item was between 2000 mg/kg bw and 5000 mg/kg bw in female Crl:(WI)BR rats. The test item was ranked into Category 5 of the Globally Harmonized Classification System (GHS) according to OECD Guideline No. 423. But not to be classified according to Regulation (EC) No 1272/2008 (CLP).
Executive summary:

The acute toxic class method was performed according to OECD 423 and Directive 2004/73/EC B.1. tris.

Starting dose was selected on the basis of the available information about the test item (Preliminary study: LD50: 2000-5000 mg/kg bw, based on sponsor’s information). The starting dose was 5000 mg/kg bw. One animal was treated with 5000 mg/kg bw dose. The first animal died, so dosing was proceeded at 2000 mg/kg bw in accordance with the flow charts in Annex 2 of the OECD guideline No. 423. Three animals were treated with 2000 mg/kg bw dose. Only one animal died in the second step at 2000 mg/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. Only one animal died in the third step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) was met.

Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals died on the treatment day, 1st and 2nd day, as well as 15th day after the treatment in survivor animals.

The LD50 value of the test item was between 2000 mg/kg bw and 5000 mg/kg bw. The test item was ranked into Category 5 of the Globally Harmonized Classification System (GHS) according to OECD Guideline No. 423, but not determined to be classfied according to Regulation (EC) No 1272/2008 (CLP).