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Description of key information

The oral LD50 of the test substance after single application to female Wistar rats is 1000 mg/kg bw (test procedure in accordance with OECD 423).

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:
2016-10-07 to 2017-01-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
December 17, 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation:
* step 1: 187 – 193 g;
* step 2: 158 – 173 g
* step 3: 163 – 174 g
- Fasting period before study: 16-19 hours
- Housing: Full barrier in an air-conditioned room
- Diet: ad libitum, free access to Altromin 1324 maintenance diet for rats and mice
- Water: ad libitum, 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)
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE (Aqua ad injectionem, AlleManPharma, expiry date: 10/2018)
- Concentration in vehicle: 0.2 g/mL for 2000 mg/kg dose (first step), 0.0312 g/mL and 0.032 g/mL for 300 mg/kg dose (second and third step)
- Amount of vehicle (if gavage): 10 mL/kg bw (feeding tube)
- Justification for choice of vehicle: this vehicle was chosen due to its non-toxic characteristics
- Lot/batch no.: 511535
- Purity: pure

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw
The test item was administered at a single dose by gavage using a feeding tube.

DOSAGE PREPARATION:
To consider purity of the active component, a correction factor of 1.04 was adopted.
The test item was weighed out into a tared plastic vial on a precision balance. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before each dose administration.
For all animals of the first step, 2.08 g of the test item was dissolved in the vehicle to gain a final volume of 10 mL and to achieve a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight.
For all animals of the second step, 0.312 g of the test item was dissolved in the vehicle to gain a final volume of 10 mL and to achieve a dose of 300 mg/kg body weight at a dose volume of 10 mL/kg body weight.
For all animals of the third step, 0.320 g of the test item was dissolved in the vehicle to gain a final volume of 10 mL and to achieve a dose of 300 mg/kg body weight at a dose volume of 10 mL/kg body weight.
Dose formulations were made shortly before each dosing occasion.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:
It is the principle of the acute toxic class method that, based on a stepwise procedure with the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test item to enable its classification. The item is tested using a stepwise procedure with up to four fixed doses. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step.
This study provides information both for hazard assessment purposes and for hazard classification purposes. Results enable compounds to be ranked in different classification systems.

Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
dose group 2000 mg/kg bw: 3;
dose group 300 mg/kg bw: 6
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days; animals were observed for general clinical signs, morbidity and mortality

- Frequency of observations and weighing: 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. 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. All abnormalities were recorded. The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.

- Necropsy of survivors performed: yes, 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 and examined macroscopically for gross pathological changes. In the absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.
Statistics:
N.A.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
1 000 mg/kg bw
Based on:
test mat.
Mortality:
The starting dose was selected to be 2000 mg/kg body weight. Compound-related mortality was recorded for 2 animals (out of 3 animals) of step 1. Based on the results and according to the acute toxic class method regime, a second step was performed at a dose of 300 mg/kg body weight. No compound-related mortality was recorded for any animal of step 2. Based on these results and according to the acute toxic class method regime, a third step was performed at a dose of 300 mg/kg body weight. No compound-related mortality was recorded for any animal of step 3. Based on these results and according to the acute toxic class method regime no further testing was required.
Clinical signs:
See Table 1 in section "Any other information on results incl. tables".
Body weight:
None of the animals showed weight loss during the observation period (see Table 2 in section "Any other information on results incl. tables").
Gross pathology:
With the exception of acut injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Clinical Signs

Table 1: Clinical Signs - Individual Data

Step

Animal No. /Sex

Starting Dose [mg/kg bw]

Time Point

Observations

1

1 /Female

2000

0 – 10 min

Moderately reduced spontaneous activity, prone position, slow movements, half eyelid closure.

10 – 60 min

Moderately reduced spontaneous activity, prone position, slow movements, severe ataxia, half eyelid closure, muscle twitches.

60 – 120 min

Moderately reduced spontaneous activity, slow movements, severe ataxia, half eyelid closure, hunched posture.

120 – 180 min

Moderately reduced spontaneous activity, hunched posture, slightly piloerection, half eyelid closure.

180 – 240 min

Slightly reduced spontaneous activity, hunched posture, slightly piloerection, half eyelid closure.

240 min – d 15

nsf

2 & 3 / Female

2000

0 – 10 min

Moderately reduced spontaneous activity, prone position, slow movements, half eyelid closure.

10 – 30/35 min

died spontaneously

 

4 – 9 / Female

300

0 min – d 15

nsf

bw = body weight; d = day (day 1 = day of administration); h = hour(s);  min = minute(s);  nsf = no specific findings

Body Weight Development

Table 2: Absolute Body Weights in g and Body Weight Change in %

Step

Animal No./ Sex

Starting Dose [mg/kg bw]

BW [g]

BW Change in comparison to Day 1 [%]

Day 1

Day 8

Day 15

Day 15

1

1 / Female

2000

187

216

210

12

2 / Female

2000

193

Died

spontaneously

-

3 / Female

2000

187

-

2

4 / Female

300

158

192

203

28

5 / Female

300

173

200

205

18

6 / Female

300

166

188

197

19

3

7 / Female

300

163

186

193

18

8 / Female

300

163

197

214

31

9 / Female

300

174

198

211

21

BW = body weight

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The oral LD50 value in rats after single oral application of 1-Benzyl-3-carbamoyl-pyridinium, chloride is 1000 mg/kg bw.
Executive summary:

In an acute oral toxicity study (OECD 423) three female Wistar rats were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg bw in a first step. In the second and third step three females each were dosed with 300 mg/kg bw. The test item was dissolved in aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg. Animals were observed for 14 days. Two out of three animals treated with the test item at a dose of 2000 mg/kg died spontaneously on the day of treatment within 30 minutes after dose administration. The third animal of this dose step and all animals treated with the test item at a dose of 300 mg/kg survived until the end of the study. While animals treated with 300 mg/kg bw did not show test-item related signs of toxicity, the most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, prone position, slow movements, ataxia, hunched posture, muscle twitches, piloerection, half eyelid closure. All symptoms recovered within up to 1 day post dose in the surviving animal. Throughout the 14-day observation period, the weight gain of the surviving animals was within the normal range of variation for this strain. At necropsy, no treatment-related macroscopic findings were observed in any animal. Based on the results the median lethal dose of the test item is 1000 mg/kg bw.

On the basis of the test results given below and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008 the substance should be classified into Category 4. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
1 000 mg/kg bw
Quality of whole database:
GLP Guideline study without deviations from the study protocol (OECD 423).

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

Data is available for the target substance. In an acute oral toxicity study (OECD 423) three female Wistar rats were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg bw in a first step. In the second and third step three female rats each were dosed orally with 300 mg/kg bw. The test item was dissolved in aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg bw. Animals were observed for 14 days. Two out of three animals treated with the test item at a dose of 2000 mg/kg died spontaneously on the day of treatment within 30 minutes after dose administration. The third animal of this dose step and all animals treated with the test item at a dose of 300 mg/kg survived until the end of the study. Based on the results, the oral LD50 can be considered to be 1000 mg/kg bw.

Justification for classification or non-classification

On the basis of the test results obtained in the OECD 423 Acute Oral Toxicity study (Acute Toxic Class Method) and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008 the substance should be classified as harmful if swallowed (H302, Acute Tox. 4).