Registration Dossier

Administrative data

Description of key information

The acute oral toxicity of the test item was determined in accordance with OECD TG 423. The LD50 is female rats was 500 mg/kg bw.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 21, 2018 - July 14, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17th December 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
December 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST rats
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 11 weeks old in all groups
- Weight at study initiation:
Body weight range at starting (first group): 174-188 g
Body weight range at starting (second group): 178-186 g
Body weight range at starting (third group): 170-181 g
Body weight range at starting (fourth group): 179-190 g
- Fasting period before study: one day
- Housing: 3 animals/sex/cage,Type II polypropylene/polycarbonate rat type cages with a solid floor, stainless steel wire covers and self-feeding baskets
- Diet: ad libitum, ssniff® SM R/M-Z+H complete diet, ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum, tap water
- Acclimation period: 26 days in first group, 27 days in second group, 28 days in third group and 29 days in fourth group

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 mg/mL (2000 mg/kg bw dose group), 30 mg/mL (300 mg/kg bw dose group)
- Justification for choice of vehicle: Test item is soluble in vehicle.

MAXIMUM DOSE VOLUME APPLIED: 10 mgL/kg bw

CLASS METHOD
- Rationale for the selection of the starting dose: The starting dose was selected on the basis of the available information about the test item.
Doses:
2000 mg/kg bw, 300 mg/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The body weight was recorded on day 0 (shortly before the treatment), on day 1, on day 2, on day 7 and on day 15 on all animals with a precision of 1 g, respectively.
Inspection for signs of morbidity and mortality were made twice daily at the beginning and end of the working day.
Animals were observed individually after dosing once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h, after the treatment and once per day for 14 days thereafter. The body weight were recorded on day 0 (shortly before the treatment), on day 7 and on day 15 on all animals with a precision of 1 g, respectively.
- Necropsy of survivors performed: yes
Statistics:
None
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
500 mg/kg bw
Based on:
test mat.
Mortality:
All female rats dosed at 2000 mg/kg bw died during the study. All animals of group 1 died on Day 2. One animal of group 2 died on Day 1 and two animals of same group died on Day 2. All deaths might be a consequence of systemic toxic effect of the test item.
No mortality occurred at 300 mg/kg bw single oral dose of the test item. All rats in step 3 and step 4 survived until the end of the 14-day observation period.
Clinical signs:
In group 1 treated with 2000 mg/kg bw clinical signs comprised of decreased activity (15 cases out of 18 observations), tremor (3/18), abnormal gait (9/18), incoordination (3/18), bedding chewing (9/18), decreased righting reflex (1/18), decreased body tone (1/18), decreased abdominal tone (1/18), closed eyes (15/18) and piloerection (15/18). Decreased activity (score -1, -2), tremor (score +2), abnormal gait (score +1, +2), incoordination (score +2), bedding chewing (score +2), closed eyes (score +1, +2), and piloerection (score +1, +2) occurred in all animals. Decreased righting reflex (score -1), decreased body tone (score -1) and decreased abdominal tone (score -1) were observed in one animal. These symptoms were detected on the treatment day between 1 and 4 hours after the treatment and on Day 1. All symptoms were regarded as test item related.
In group 2 treated with 2000 mg/kg bw clinical signs comprised of decreased activity (15 cases out of 17 observations), tremor (2/17), incoordination (1/17), bedding chewing (9/17), closed eyes (14/17) and piloerection (14/17). Decreased activity (score -1, -2, -3), bedding chewing (score +2), closed eyes (score +1, +2, +3) and piloerection (score +1, +2, +3) occurred in all animals. Tremor (score +2) was observed in two animals. Incoordination (score +2) was detected in one animal These symptoms were detected on the treatment day between 30 minutes and 4 hours after the treatment and on Day 1. All symptoms were regarded as test item related.
No treatment related symptoms were observed in the 300 mg/kg bw test item dose groups (group 3 and 4) throughout the 14-day post-treatment period.
Body weight:
The mean body weight and body weight gain data of group 1 (2000 mg/kg bw) and of group 2 (2000 mg/kg bw) could not be evaluated, because all rats died.
In group 3 and 4 (300 mg/kg bw) the mean body weight and body weight gain of the animals corresponded to their species and age throughout the study.
Gross pathology:
All rats treated with 2000 mg/kg bw died spontaneously during the study and were necropsied on Day 1 or on Day 2, as well. All animals treated with 300 mg/kg dose of test item survived until the scheduled necropsy on Day 15.
Internal macroscopic changes like not differentiated kidneys medulla and reddish-brown kidneys were recorded in all animals of group 1 and group 2. Haemorrhages in the stomach lining were observed in all animals of group 1 and in two animals of group 2. Mucous fur on the organs was found in two animals of group 1 and in all animals of group 2. Besides, reddish mucous membrane of the stomach was observed in one animal of group 2. These internal macroscopic changes observed were regarded as test item related.
Severe hydrometra was found in two animals in group 3 and in one animal of group 4. Moderate hydrometra was detected in one animal of group 3 and in of group 4 (300 mg/kg bw). Hydrometra is a physiological finding and connected to the estrous cycle of the animal.
No pathological changes were found related to the effect of the test item during the macroscopic examination of animals of group 3 and of group 4.

Table 1: Results

 Dose (mg/kg bw)  Mortality (dead/treated)  LD50 (mg/kg bw)  CLP category
 2000  3/3 (at 1st step)     500 Category 4    
 300  0/6
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
For this acute oral toxicity study with the test item in rats the determined LD50 is 500 mg/kg bw.
Executive summary:

An acute oral toxicity study was carried out using the class method according to OECD TG 423. The method was conducted using a stepwise procedure with 2000 mg/kg bw as the starting dose in three female rats. The starting dose was selected on the basis of the available information about the test item.

No animal died in the first step by Day 1, so further three female rats were treated with the same (2000 mg/kg bw) dose. All animals died in the first and second step between Day 1 and Day 2, so further three female rats were treated with the lower (300 mg/kg bw) dose. No animal died in third step, so further three animals were treated with the same dose. No animal died in fourth step, too. The test was finished as a stopping criterion of Annex 2d of OECD Guideline No. 423 was met.

Animals were weighed and observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals that died on Day 1 and on Day 2, as well as in animals that survived until the end of the observation period on Day 15.

Lethality, Clinical symptoms and Body weight:

All six animals treated with 2000 mg/kg bw test item died between Day 1 and Day 2. No lethality was noted at a single oral dose of 300 mg/kg bw.

In group 1, at a dose level of 2000 mg/kg bw, CNS-symptoms and abnormal movement (decreased activity, tremor, bedding chewing, closed eyes), disturbances of the coordination (abnormal gait, incoordination), decreased righting reflex, decreased muscular tension (abdominal- and body tone) and disturbance of the autonomic functions (piloerection) were observed in animals. These symptoms were detected on the treatment day between 1 and 4 hours after the treatment and on Day 1.

In group 2, at a dose level of 2000 mg/kg bw, CNS-symptoms and abnormal movement (decreased activity, tremor, bedding chewing, closed eyes), disturbance of the coordination (incoordination) and disturbance of the autonomic functions (piloerection) were observed in animals. These symptoms were detected on the treatment day between 30 minutes and 4 hours after the treatment and on Day 1.

In group 3 and 4, at a dose level of 300 mg/kg bw no clinical symptoms were observed on the day of the treatment and during the 14-day observation period. The general state and behaviour of all experimental animals were normal.

The body weight development was normal in all surviving animals.

Gross pathology:

Altogether 6 animals died, and 6 animals were sacrificed according to schedule at the end of the study period.

Internal findings included mucous fur on the organs, haemorrhages in the stomach lining, not differentiated kidneys medulla, reddish-brown kidneys and reddish mucous membrane of the stomach.

All of the animals treated with 300 mg/kg bw proved to be free of treatment related gross pathological changes.

Evaluation:

The method used is not intended to allow for the calculation of a precise LD50 value. However for this acute oral toxicity study with the test item in rats the determined LD50 is 500 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
500 mg/kg bw
Quality of whole database:
The quality of the study is considered sufficient for assessment.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An acute oral toxicity study was carried out using the class method according to OECD TG 423. The method was conducted using a stepwise procedure with 2000 mg/kg bw as the starting dose in three female rats. The starting dose was selected on the basis of the available information about the test item.

No animal died in the first step by Day 1, so further three female rats were treated with the same (2000 mg/kg bw) dose. All animals died in the first and second step between Day 1 and Day 2, so further three female rats were treated with the lower (300 mg/kg bw) dose. No animal died in third step, so further three animals were treated with the same dose. No animal died in fourth step, too. The test was finished as a stopping criterion of Annex 2d of OECD Guideline No. 423 was met.

Animals were weighed and observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out in animals that died on Day 1 and on Day 2, as well as in animals that survived until the end of the observation period on Day 15.

Lethality, Clinical symptoms and Body weight:

All six animals treated with 2000 mg/kg bw test item died between Day 1 and Day 2. No lethality was noted at a single oral dose of 300 mg/kg bw.

In group 1, at a dose level of 2000 mg/kg bw, CNS-symptoms and abnormal movement (decreased activity, tremor, bedding chewing, closed eyes), disturbances of the coordination (abnormal gait, incoordination), decreased righting reflex, decreased muscular tension (abdominal- and body tone) and disturbance of the autonomic functions (piloerection) were observed in animals. These symptoms were detected on the treatment day between 1 and 4 hours after the treatment and on Day 1.

In group 2, at a dose level of 2000 mg/kg bw, CNS-symptoms and abnormal movement (decreased activity, tremor, bedding chewing, closed eyes), disturbance of the coordination (incoordination) and disturbance of the autonomic functions (piloerection) were observed in animals. These symptoms were detected on the treatment day between 30 minutes and 4 hours after the treatment and on Day 1.

In group 3 and 4, at a dose level of 300 mg/kg bw no clinical symptoms were observed on the day of the treatment and during the 14-day observation period. The general state and behaviour of all experimental animals were normal.

The body weight development was normal in all surviving animals.

Gross pathology:

Altogether 6 animals died, and 6 animals were sacrificed according to schedule at the end of the study period.

Internal findings included mucous fur on the organs, haemorrhages in the stomach lining, not differentiated kidneys medulla, reddish-brown kidneys and reddish mucous membrane of the stomach.

All of the animals treated with 300 mg/kg bw proved to be free of treatment related gross pathological changes.

Evaluation:

The method used is not intended to allow for the calculation of a precise LD50 value. However for this acute oral toxicity study with the test item in rats the determined LD50 is 500 mg/kg bw.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is classified for acute oral toxicity Cat 4 (H302) according to Regulation (EC) No 1272/2008 (CLP), as amended for the 12th time in Regulation (EU) No 2019/521.