Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-06 to 2016-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
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: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
WI(Han)
Sex:
female
Details on test animals and environmental conditions:
Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test item was administered at a single dose by gavage using a feeding tube in the morning:
Step 1: 09:00 a.m.
Step 2: 08:15 a.m.
Step 3: 07:00 a.m.
The test item was administered at a dose volume of 10 mL/kg body weight.
Doses:
- step 1: 300 mg/kg body weight
- step 2: 50 mg/kg body weight
- step 3: 50 mg/kg body weight.
No. of animals per sex per dose:
- steps 1 to 3: 3 females each dose
Control animals:
no
Details on study design:
Preparation of the animals
The animals were marked for individual identification by tail painting. Prior to the administration a detailed clinical observation was made of all animals. Only healthy animals were used. Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test item was administered. Food was provided again approximately 4 hours post dosing.

Observation Period
The surviving animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality.

Weight Assessment
The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.

Clinical Examination
A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded. Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology
Animals sacrificed for ethical reasons during the observation period were necropsied as soon as they were killed.
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw. All animals were subjected to gross necropsy. All gross pathological changes were recorded but not preserved for possible histopathological evaluation.

Evaluation of Results
Results were interpreted according to OECD Guideline 423, Annex 2 and GHS. Individual reactions of each animal were recorded at each time of observation. Toxic response data were recorded by dose level. Nature, severity and duration of clinical observations were described. The body weight changes were summarised in a tabular form. Necropsy findings were described.
Statistics:
n.a.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
200 mg/kg bw
Based on:
test mat.
Mortality:
- At a concentration of 300 mg/kg all animals had to be sacrificed within the first hours after dose administration.
- At a concentration of 50 mg/kg one of six animals died within the first five hours after dose administration.
Clinical signs:
The test item showed mortality and other acute oral toxicity characteristics after a single dose administration. Animals exhibited moderately reduced spontaneous activity, opisthotonos, severe ataxia, moderate piloerection, cyanosis and half eyelid closure within the first four hours after application of the test item.
Body weight:
None of the animals showed weight loss during the observation period
Gross pathology:
All animals treated with 300 mg/kg bw showed a bloody stomach, duodenum, jejunum, ileum and peyer’s patches, one animal treated with 50 mg/kg bw showed bloody stomach, duodenum, jejunum, ileum and peyer’s patches.

Any other information on results incl. tables

LD50 Cut-Off

Starting Dose (mg/kg bw) Number of animals Number of intercurrent deaths LD50 Cut-Off (mg/kg bw)
300 3 3 200
50 6 1

bw = body weight

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat): 200 mg/kg bw.
Executive summary:

The purpose of this study was to assess the toxicity of the test article when administered to rats after a single oral dose. The study was carried out in accordance with OECD TG 423 and in compliance to GLP.

One group, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 300 mg/kg body weight. The test item was dissolved in aqua ad injectionem (sterile water) at a concentration of 0.03 g/mL and administered at a dose volume of 10 mL/kg. Two further groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 50 mg/kg body weight. The test item was suspended with the vehicle aqua ad injectionem (sterile water) at a concentration of 0.005 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at the test facility were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

All animals treated with the test item at a dose of 300 mg/kg had to be sacrificed for ethical reasons on test day 1. One animal treated with the test item at a dose of 50 mg/kg died spontaneously on the day of treatment. All other animals treated with the test item at a dose of 50 mg/kg survived until the end of the study showing test-item related signs of toxicity.

The most relevant clinical findings in the animals treated with the test item at a dose of 300 mg/kg bw were reduced spontaneous activity, prone position, ataxia, slow movements, piloerection, recumbency, opisthotonos, cyanosis and half eyelid closure.

The most relevant clinical findings in the animals treated with the test item at a dose of 50 mg/kg bw were reduced spontaneous activity, hunched posture, prone position, ataxia, piloerection, abnormal breathing, eyes closed and half eyelid closure. All symptoms recovered within up to 1 day post-dose.

Macroscopic findings of animals not having survived until the end of the observation period: Necropsy revealed a bloody stomach and a bloody intestine in all animals (No. 1-3, 9). Macroscopic findings of surviving animals: At necropsy, no treatment-related macroscopic findings were observed in any animal (no. 4-6, 7+8).

Under the conditions of the present study, a single oral application of the test item to rats at a dose of 300 mg/kg body weight was associated with signs of toxicity or mortality. The median lethal dose of the test item after a single oral administration to female rats, observed

over a period of 14 days is: LD50 cut-off (rat): 200 mg/ kg bw.