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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 May 2016 to 02 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Amber liquid
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
EXPERIMENTAL ANIMALS
- Species and strain: Sprague Dawley rats
- Source: Charles River Laboratories, Research Models and Services GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany.
- Sex: Female, nulliparous and non-pregnant.
- Age of animals at dosing: Young healthy adult rats, 8 weeks old.
- Body weight at treatment: 189 to 200 g
- Acclimatisation period: 6 or 7 days.

HUSBANDRY
- Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
- Number of animal room: 522/12.
- Housing: Three animals per cage.
- Cage type: Type II polypropylene/polycarbonate.
- Bedding: Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study.
A copy of the Certificate of Analysis is retained in the archive at CiToxLAB Hungary Ltd.
- Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 20.1 to 23.7 °C.
- Relative humidity: 36 to 70 %.
- Temperature and relative humidity were measured continuously and recorded twice daily during the study.
- Ventilation: 15 to 20 air exchanges/hour.
- Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.

ANIMAL IDENTIFICATION
- Animals were individually identified using numbers written on the tail with an indelible
marker pen. The numbers were given on the basis of CiToxLAB Hungary Ltd.'s Master File, for each animal allocated to the treatment groups.
- Cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
Sigma Aldrich (batch MKBQ0728V; expiry date 30 September 2016; room temperature storage)
Details on oral exposure:
FORMULATION
- The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of CiToxLAB Hungary Ltd. on the day of administration.
- The formulation container was stirred continuously until treatment was completed.

Doses:
2000 mg/kg bw
No. of animals per sex per dose:
Two groups of three animals
Control animals:
no
Details on study design:
FOOD AND WATER SUPPLY
- Animals received ssniff SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (Batch No.: 278 5652, expiry date: November 2016), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 ml bottles, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József, A.u.36., Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd.

TEST ITEM ADMINISTRATION
- The initial dose level was selected by the study director to be that which is most likely to produce mortality in some of the dosed animals. In the lack of any preliminary toxicological information, 2000 mg/kg bw was selected to be the starting dose.
- Initially, three female animals (Group 1) were treated with 2000 mg/kg bw of test item. No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 mg/kg bw. As no mortality was observed in this second dose group, further testing was not required according to the test guidelines (OECD 423, Commission Regulation (EC) NO 440/2008 of 30 May 2008, B.1.Tris).
- A single oral gavage administration was followed by a fourteen-day observation period. On the night before treatment, the animals were fasted. The food but not water was withheld during an overnight period. Animals were weighed just before treatment. The test item was administered by oral gavage in the morning. Food was returned three hours after treatment.

CLINICAL OBSERVATIONS
- Clinical observations were performed on all surviving animals at 0.5, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter.
- Individual observations were performed on the skin, fur, eyes, mucous membranes, 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 MEASUREMENT
- The body weights of the animals were recorded on the day before treatment (Day -1), on the day of the treatment (Day 0) and weekly thereafter.

NECROPSY
- Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthanimal 40%; Lot No.: 1409236-06, Expiry Date: September 2017, produced by: AlfasanNederland BV, Woerden, Netherlands).
- After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed. Macroscopic abnormalities were recorded.
Statistics:
EVALUATION OF TEST RESULTS
- The method used was not intended to allow the calculation of a precise LD50 value.
- Clinical signs, body weight, body weight gain and gross macroscopic data were tabulated.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
- The test item did not cause mortality at a dose level of 2000 mg/kg bw.
Clinical signs:
- Treatment with the test item at a dose level of 2000 mg/kg bw caused decreased activity (4/6), hunched back (6/6), piloerection (6/6), and increased salivation (1/6) (see Appendix 1, attached).
Body weight:
- Body weight gains during the study showed no indication of a test item-related effect (see Appendix 2, attached).
Gross pathology:
- There was no evidence of the macroscopic effects at a dose level of 2000 mg/kg bw (see Appendices 3 and 4, attached).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Sprague Dawley rat was found to be > 2000 mg/kg bw.
Executive summary:

GUIDELINE

The single-dose oral toxicity of the test item was investigated according to the acute toxic class method (OECD 423, Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.Tris and OPPTS 870.1100) in Sprague Dawley rats.

 

METHODS

Two groups of three female Sprague Dawley rats were treated with the test item at a dose level of 2000 mg/kg bw (Group 1 and Group 2). A single oral treatment was carried out by gavage for each animal after withdrawal of food overnight. Food was made available again 3 hours after the treatment. The test item was administered formulated in peanut oil at a concentration of 200 mg/mL at a dose volume of 10 mL/kg bw. Initially, three female animals (Group 1) were treated with 2000 mg/kg bw of test item. No mortality was observed, therefore a further three animals were treated at the dose level of 2000 mg/kg bw. No mortality was observed in the confirmatory group; therefore no further testing was required according to the test guidelines. Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and before necropsy. All animals were subjected to a necropsy and a macroscopic examination.

 

RESULTS

The test itemdid not cause mortality at a dose level of 2000 mg/kg bw. Clinical observations at the dose level of 2000 mg/kg bw were reported as decreased activity (4/6), hunched back (6/6), piloerection (6/6), and increased salivation (1/6). Body weight gains of treated animals during the study did not indicate a test item-related effect. No macroscopic observations were reported at 2000 mg/kg bw.

 

CONCLUSION

The acute oral median lethal dose (LD50) of the test item in the female Sprague Dawley rat was found to be > 2000 mg/kg bw.