Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 26, 2016 to September 28, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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
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
Details on test material:
Purity >99.1 %; Appearance: white powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Species and strain: Crl: WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH
Hygienic level at arrival: SPF
Hygienic level during the study: standard housing conditions
Number of animals: 9 animals, 3 animals/group
Sex: Female, nulliparous and non-pregnant.
Age of animals at dosing: young healthy adult rats, 11 weeks old
Body weight at treatment: 216 – 255 g
Acclimatization period: 27 or 28 or 29 days

Husbandry
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 242/2
Housing: 3 animals / cage
Cage type: type II polypropylene/polycarbonate
Bedding: Lignocel 3/4-S Hygienic Animal Bedding” produced by J. Rettenmaier & Söhne GmbH and certified nest building material were available to animals during the study.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 26.0 °C
Relative humidity: 26 – 69 %
Ventilation: 15 – 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.
The temperature and relative humidity were measured continuously and recorded twice daily during the study.

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, ad libitum, and tap water from the municipal supply, as for human consumption from a 500 ml bottles, ad libitum. The food is 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.

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. The 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:
methylcellulose
Remarks:
1 %; dose volume: 10 mL/kg bw
Details on oral exposure:
The test substance was administered by oral gavage formulated in 1 % methyl cellulose at a concentration of 200 or 30 mg/mL at a dose volume of 10 mL/kg bw.
Doses:
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.

Group 1: 2000 mg/kg bw
Group 2: 300 mg/kg bw
Group 3: 300 mg/kg bw
No. of animals per sex per dose:
9 animals, 3 animals/group
Control animals:
no
Details on study design:
Formulation
The test substance was freshly formulated at a concentration of 200 or 30 mg/mL in the vehicle on the day of administration. The formulation container was stirred continuously up to finishing the treatment.

Dose
Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw of the test substance. The test substance caused mortality in this group in 3/3 animals and a second group (Group 2) was treated at a dose level of 300 mg/kg bw. No mortality was observed at this dose group, therefore a confirmatory group (Group 3) was treated at a dose level of 300 mg/kg bw. No mortality was observed in the confirmatory group, therefore no further testing was required.

Procedure
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 substance was administered by oral gavage in the morning. The food was returned 3 hours after the treatment.

OBSERVATIONS

Clinical Observations
Clinical observations were performed on all surviving animals at 30 minutes, 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. Moreover, the body weight of found dead animals was recorded on the day of death or at necropsy.

NECROPSY
Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthanimal 40 %). 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.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
not measured/tested
Mortality:
The test substance caused mortality at the dose level of 2000 mg/kg bw in all animals (3/3) on Day 0.
Clinical signs:
In all animals, treated with the test substance at the dose level of 2000 mg/kg bw decreased activity (3/3), hunched back (3/3), prone position (3/3) and incoordination (3/3) was observed.
In all animals, treated with the test substance at the dose level of 300 mg/kg bw no clinical signs were noted during the observation period.
Body weight:
There were no treatment related effects on body weight or body weight gain during the observation period on the surviving animals (300 mg/kg bw).
Gross pathology:
In the rats, dosed at 2000 mg/kg bw, that were found dead, the following observations were made:
- Presence of diffuse or multifocal, dark red discoloration in the non-collapsed lungs
- Presence of white, foamy material in the trachea in 2/3 animals
These findings were considered to be test substance related.
There was no evidence of any gross findings in the rats, at a dose level of 300 mg/kg bw, necropsied at the end of the observation period on day 14.

Any other information on results incl. tables

The method used did not allow the calculation of a precise LD50 value. The test substance was ranked into categories of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 (Annex 2d).

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the study conditions, the acute oral LD50 value of the test substance was found to be between 300 and 2000 mg/kg bw in female Crl:WI Wistar. According the GHS criteria, the test substance can be ranked as "Category 4" for acute oral exposure.
Executive summary:

A study was conducted to determine the potential toxic effect of the test substance when administered as a single oral dose to Crl:WI Wistar rats according to OECD Guideline 423 Acute Toxic Class (ATC) method and EU Method B.1, in compliance with GLP. A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test substance was administered formulated in 1% methyl cellulose at a concentration of 200 or 30 mg/mL at a dose volume of 10 mL/kg bw. Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. The test substance caused mortality in this group in all animals. The second group (Group 2) was treated at a dose level of 300 mg/kg bw. No mortality was observed, therefore a confirmatory group (Group 3) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required. Clinical observations were performed on the surviving animals 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. Moreover, the body weight of found dead animals was recorded on the day of death or at necropsy. All animals were subjected to a necropsy and a macroscopic examination. The test substance caused mortality at the dose level of 2000 mg/kg bw on day 0 in all animals. Treatment at this dose level caused first decreased activity, hunched back, prone position and incoordination in all animals. The following macroscopic treatment-related observations were made: presence of diffuse or multifocal, dark red discoloration in the non-collapsed lungs, presence of white, foamy material in the trachea in 2/3 animals. Treatment at the dose level of 300 mg/kg bw did not cause any clinical signs during the observation period. No treatment related effects on body weight or body weight gain were noted. There was no evidence of any gross findings in the rats, at a dose level of 300 mg/kg bw, necropsied at the end of the observation period on day 14. Under the study conditions, the acute oral LD50 value of the test substance was found to be between 300 and 2000 mg/kg bw in female Crl:WI Wistar rats (Váliczkó, 2017).