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Description of key information

In an acute oral toxicity study according to OECD 425 in female Wistar rats, the toxicological profile of 2 -amino-5 -chloro-N,3 -dimethylbenzamide was assessed and the median lethal dose determined to LD50 = 310.2 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:
30 January 2020 to 30 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Version / remarks:
October 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:
up-and-down procedure
Limit test:
yes
Species:
rat
Strain:
other: RccHan: WIST
Sex:
female
Details on test animals and environmental conditions:
Species: Rat (Rattus norvegicus)
Strain: RccHan: WIST
Age/Weight at Dosing: 10 to 12 weeks, Weight (g) Minimum: 205.7, Maximum: 229.4
Source: Animal Breeding Facility, Jai Research Foundation
Total Number of Animals Used: Six females (nulliparous and non-pregnant)

Acclimatisation: 6 to 17 days

Animal Identification: Each rat was uniquely numbered on the tail using a tattoo machine on day 1 of acclimatisation. Appropriate labels were attached to the cages indicating the study number, test item code, group number, sex, dose, type of study, cage number and animal number.

Caging: Polypropylene rat cages covered with stainless-steel grid top were used. Autoclaved clean rice husk was used as the bedding material. Wooden blocks were provided as enrichment material.

Water Bottle: Each cage was supplied with a polypropylene water bottle with a stainless-steel nozzle.
Housing: Maximum of three rats per cage, Room No. BMR 28

Room Sanitation. daily:
1. Rack was cleaned with cloth,
2. Floor of experimental procedure room was swept,
3. All work tops and the floor were mopped with a disinfectant solution (Dettol 2.5%).

Feed and Water
The rats were provided with feed and water, ad libitum. The quality of feed and water is regularly monitored at Jai Research Foundation. There were no known contaminants in the feed and water at levels that would have interfered with the experimental results obtained.
Feed: Teklad certified Global High Fiber Rat/Mice Feed manufactured by Envigo, USA.
Water: UV sterilised water filtered through Reverse Osmosis water filtration system.

Environmental Conditions
Animal Room: BMR 28, Department of Toxicology
Temperature: 20 to 23°C
Relative Humidity: 56 to 66%
Air Changes: Minimum 15 air changes/hour
Photoperiod : The photoperiod was 12 hours artificial light and 12 hours darkness, light hours being 06:00 h–18:00 h (photoperiod was maintained through automatic timer)


Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: Individual dose volume was adjusted according to body weight and dose level (10 mL/kg body weight):
Dose: 175 mg/kg = Dose Volume administered: 2.06–2.25 mL; Dose: 550 mg/kg = Dose Volume administered: 2.17–2.29 mL
- Amount of vehicle: a constant volume of 10 mL/kg body weight

ORAL GAVAGE
- All rats were dosed by oral gavage (day 0) using a metal cannula attached to a BD 1 mL disposable syringe, which was graduated up to 1 mL. Rats were fasted overnight, prior to dosing, until three hours post-dosing.
Doses:
Dose: 175 mg/kg = Dose Volume administered: 2.06–2.25 mL;
Dose: 550 mg/kg = Dose Volume administered: 2.17–2.29 mL
No. of animals per sex per dose:
three animals/female/Dose: 175 mg/kg
three animals/female/Dose: 550 mg/kg
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: At 0.5, 1, 2, 3, 4 and 5 h post-administration on the day of dosing.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: The clinical signs were recorded at least once a day. Individual body weight was recorded prior to dosing on day 0, and on days 7 and 14 and at death.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
310.2 mg/kg bw
Based on:
test mat.
95% CL:
> 175 - <= 550
Mortality:
Mortality was observed in rats treated at 550 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight (rat Nº 2, 4, and 6), while, no mortality was observed in rats treated at 175 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight (rat N˚ 1, 3, and 5).
Clinical signs:
Clinical sign (lethargy) was observed in rats treated at 550 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight (rat Nº 2, 4, and 6), while no clinical sign was observed in rats treated at 175 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight (rat N˚ 1, 3, and 5).
Body weight:
A normal gain in the body weight was observed in surviving rat treated at 175 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight (rat N˚ 1, 3, and 5).
Gross pathology:
External Examination:
An external examination of the found dead and terminally sacrificed rats did not reveal any abnormality.
Internal Examination:
An internal examination of the found dead and terminally sacrificed rats did not reveal any lesion.

Dose, Mortality/Rats Treated:

Dose (mg/kg body weight)

Female rats (mortality/total)

175

0/3

550

3/3

Test Sequence and Mortalities:

Rat N°

Dose

(mg/kg body weight)

Mortality after Dosing

½ - 5 h

24 h

48 h

72 h

4 – 7

Day

8 – 14

Day

1

175

O

O

O

O

O

O

2

550

O

O

X

-

-

-

3

175

O

O

O

O

O

O

4

550

O

X

-

-

-

-

5

175

O

O

O

O

O

O

6

550

O

O

O

X

-

-

Note: O = Survived, X = Dead, - = Not applicable.

Individual Clinical Observations:

Rat

Dose (mg/kg body weight)

Clinical Signs Observed after Dosing

At Hour

(Day 0)

On Day

0.5

1

2

3

4

5

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1

175

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

550

1

1

1

1

11

11

11

2

-

-

-

-

-

-

-

-

-

-

-

-

3

175

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

4

550

1

1

1

1

11

11

11, 2

-

-

-

-

-

-

-

-

-

-

-

-

-

5

175

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

6

550

1

1

11

11

11

11

11

11

2

-

-

-

-

-

-

-

-

-

-

-

Note: Day 0 = Day of dosing, - = Not applicable.

Clinical Sign: 1 = Normal, 2 = Dead, 11= Lethargy.

Individual Necropsy Findings:

Rat N°

Dose (mg/kg body weight)

Mode of Death

External

Internal

1

175

Terminal sacrifice

No abnormality detected

No abnormality detected

2

550

Found dead

No abnormality detected

No abnormality detected

3

175

Terminal sacrifice

No abnormality detected

No abnormality detected

4

550

Found dead

No abnormality detected

No abnormality detected

5

175

Terminal sacrifice

No abnormality detected

No abnormality detected

6

550

Found dead

No abnormality detected

No abnormality detected

 

Interpretation of Results

The LD50 was calculated as per the Dixon’s maximum likelihood method using software (AOT 425 StatPgm) and found to be 310.2 mg/kg body weight in female Wistar rats with 95% confidence interval is 175 to 550 mg/kg body weight.

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based on results of this study, the acute oral median lethal dose for 2-amino-5-chloro-N,3-dimethylbenzamide in Wistar rats was found to be 310.2 mg/kg body weight in female Wistar rats with 95% confidence interval is 175 to 550 mg/kg body weight.
Executive summary:

In an acute oral toxicity study in female Wistar rats (10 to 12 weeks old), the toxicological profile of 2-amino-5-chloro-N,3-dimethylbenzamide was assessed and the median lethal dose determined. A single oral dose of 2-amino-5-chloro-N,3-dimethylbenzamide was administered orally to fasted rats (formulated using corn oil as a vehicle and at a constant dose volume of 10 mL/kg) through gavage. Initially rat N° 1 was tested with a starting dose-level of 175 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight. The tested rat survived at this dose level, subsequently five additional female Wistar rats received dose of 550 mg (rat N˚ 2, 4 and 6) and 175 mg (rat N˚ 3 and 5) 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight, according to the Up and Down Procedure. All surviving rats were observed for 14 days.

Mortality was observed in rats treated at 550 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight while no mortality was observed in rats treated at 175 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight.

Clinical sign (lethargy) was observed in rats treated at 550 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight, while no clinical sign was observed in rats treated at 175 mg 2-amino-5-chloro-N,3-dimethylbenzamide/kg body weight.

A normal gain in the body weight was observed in surviving rat.

 

The number of animals which died or showed evident toxicity is shown below:

Dose
(mg/kg bw)

Mortality
(# dead/total)

Time range of deaths (h)

Evident toxicity 
(#/total)

175

0/3

-

0/3

550

3/3

24 to 72

3/3

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
310.2 mg/kg bw

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

Justification for classification or non-classification

According to OECD test 425 provided with an median lethal dose of LD50 = 310.2 mg/kg bw by oral administration the registrant classifies 2-amino-5-chloro-N,3-dimethylbenzamide as acute toxic cat. 4 (oral) with hazard phrase H302 (harmful if swallowed).