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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 December 2017 - 25 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
EC Number:
231-034-6
EC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Cas Number:
7417-99-4
Molecular formula:
C19H20N4O2
IUPAC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan: WIST
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 11 weeks
- Weight at study initiation: 166.9 to 188.5 g
- Fasting period before study: overnight prior to dosing until three hours post-dosing
- Housing: 3 females in polypropylene rat cages covered with stainless steel grid top. Autoclaved clean rice husk was used as the bedding material. Wooden blocks were provided as enrichment material.
- Diet (e.g. ad libitum): Ad libitum. Teklad Certified Global High Fiber Rat/Mice Feed manufactured by Envigo, USA. The quality of feed is regularly monitored at Jai Research Foundation.
- Water (e.g. ad libitum): Ad libitum. UV sterilized water filtered through Reverse Osmosis water filtration system. The quality of water is regularly monitored at Jai Research Foundation.
- Acclimation period: 6 to 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23°C
- Humidity (%): 49 to 66%
- Air changes (per hr): At least 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light (06.00 to 18.00 h) and 12 hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 mg/mL and 200 mg/mL
- Amount of vehicle (if gavage): 10 mL
- Justification for choice of vehicle: recommended by OECD 423 guideline

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: without preliminary information, the selected starting dose is 300 mg/kg body weight.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
Set I (300 mg/kg bw): 3 animals; Set II (300 mg/kg bw): 3 animals; Set III (2000 mg/kg bw): 3 animals
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: toxicity and mortality at 30 min, 1h, 2 h, 3h, 4h, 6 h after adminitration. Morbidity and mortality twice a day and clinical sings daily during 14 days. Weighing= D0 (prior to dosing) then on D7, and D14.
- Necropsy of survivors performed: yes. At termination, macroscopic observation was observed.
- Other examinations performed:
Clinical observations: evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects and central nervous system effects, behavioural pattern, somatomotor activity and observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
ca. 500 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed in set I and set II treated at the dose level of 300 mg/kg bw.
All the rats were found dead in set III treated at the dose level of 2000 mg/kg bw.
Clinical signs:
No clinical sign was observed in rats treated with 300 mg/kg bw.
Clinical sign like lethargy was observed in all the rats treated with 2000 mg/kg bw.
Body weight:
Normal gain in body weight was observed on days 7 and 14 in rats treated with 300 mg/kg bw while decrease in body weight was observed on day 7 in rats treated with 2000 mg/kg bw.


Gross pathology:
External examination of found dead and terminally sacrificed rats did not reveal any abnormality.
Visceral examination of found dead rats revealed pale liver (rats treated with 2000 mg/kg bw) whereas terminally sacrificed rats did not reveal any lesion.
Lesion observed in the found dead rats could be correlated with the test item used in the present study.

Any other information on results incl. tables

Table 1: Mortality

Dose

(mg/kg body weight)

Set

Number of Rats Used

Mortality after Dosing

At Hour

On Day

0.5 – 4

6

1

2

3

4 - 7

8 - 14

300

I

3

0

0

0

0

0

0

0

II

3

0

0

0

0

0

0

0

2000

III

3

0

0

0

0

0

0

3

Table 2: Individual and Mean Body Weight (g) and Body Weight Change (%)

Dose

(mg/kg body weight)

Rat N°

Date and Time of Dosing

Volume of Test Item Administered (mL)

Body Weight (g) on Day

Percent Body Weight Change on Day

0

7

14

At Death

7

14

300

1

December 07, 2017

and

10:00 am

1.67

166.9

170.4

207.1

-

2.1

24.1

2

1.71

170.8

181.3

193.2

-

6.1

13.1

3

1.73

173.3

185.6

200.5

-

7.1

15.7

Mean

170.3

179.1

200.3

-

5.1

17.6

Standard Deviation

(±)

3.2

7.8

7.0

-

2.6

5.7

4

December 11, 2017

and

10:30 am

1.88

187.7

199.3

209.4

-

6.2

11.6

5

1.89

188.5

191.9

196.3

-

1.8

4.1

6

1.86

186.4

195.9

204.5

-

5.1

9.7

Mean

187.5

195.7

203.4

-

4.4

8.5

Standard Deviation

(±)

1.1

3.7

6.6

-

2.3

3.9

2000

7

December 13, 2017

and

10:30 am

1.82

181.7

154.9

-

134.1

-14.7

-

8

1.85

185.1

155.7

-

132.3

-15.9

-

9

1.80

180.2

151.5

-

130.6

-15.9

-

Mean

182.3

154.0

-

-

-15.5

-

Standard Deviation

(±)

2.5

2.2

-

-

0.7

-

Key: Day 0 = Before dosing , - = Not applicable

Table 3: Individual clinical observations

Set I                                                                                                                          

Dose

(mg/kg body weight)

Rat N°

Clinical Signs Observed Post-dosing

At Hour (Day 0)

0.5

1

2

3

4

6

300

1

1

1

1

1

1

1

2

1

1

1

1

1

1

3

1

1

1

1

1

1

 

Dose

(mg/kg body weight)

Rat N°

Clinical Signs Observed Post-dosing

On Days

1

2

3

4

5

6

7

8

9

10

11

12

13

14

300

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

3

1

1

1

1

1

1

1

1

1

1

1

1

1

1

 

Set II                                                                                                                           

Dose

(mg/kg body weight)

Rat N°

Clinical Signs Observed Post-dosing

At Hour (Day 0)

0.5

1

2

3

4

6

300

4

1

1

1

1

1

1

5

1

1

1

1

1

1

6

1

1

1

1

1

1

 

Dose

(mg/kg body weight)

Rat N°

Clinical Signs Observed Post-dosing

On Days

1

2

3

4

5

6

7

8

9

10

11

12

13

14

300

4

1

1

1

1

1

1

1

1

1

1

1

1

1

1

5

1

1

1

1

1

1

1

1

1

1

1

1

1

1

6

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Set III                                                                                                                                                                   

Dose

(mg/kg body weight)

Rat N°

Clinical Signs Observed Post-dosing

At Hour (Day 0)

0.5

1

2

3

4

6

2000

7

1

1

1

1

1

1

8

1

1

1

1

1

1

9

1

1

1

1

1

1

 

Dose

(mg/kg body weight)

Rat N°

Clinical Signs Observed Post-dosing

On Days

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

7

1

1

1

1

1

1

1

11

11

11, 2

-

-

-

-

8

1

1

1

1

1

11

11

11

11

2

-

-

-

-

9

1

1

1

1

1

1

1

11

11

11, 2

-

-

-

-

Clinical Signs: 1 = Normal, 2 = Dead, 11 = Lethargy

Note: Day 0 = Day of dosing, - = Not Applicable

Table 4: Individual necropsy findings

Dose

(mg/kg body weight)

Set

Rat

Mode of Death

External

Internal

300

I

1

Terminal sacrifice

No abnormality detected

No abnormality detected

2

Terminal sacrifice

No abnormality detected

No abnormality detected

3

Terminal sacrifice

No abnormality detected

No abnormality detected

II

4

Terminal sacrifice

No abnormality detected

No abnormality detected

5

Terminal sacrifice

No abnormality detected

No abnormality detected

6

Terminal sacrifice

No abnormality detected

No abnormality detected

2000

III

7

Found dead

No abnormality detected

Liver:Pale (1+)

8

Found dead

No abnormality detected

Liver:Pale (2+)

9

Found dead

No abnormality detected

Liver:Pale (+)

Key: + = Minimal, 1+ = Mild, 2+ = Moderate

Applicant's summary and conclusion

Interpretation of results:
other: Category 4 (CLP Regulation EC no. 1272/2008)
Conclusions:
The LD50 of the test item is lower than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off may be considered to be 500 mg/kg body weight by oral route in the rat.

Executive summary:

The acute oral toxicity of the test item has been tested in accordance with OECD Test Guideline 423, following GLP. 9 female Wistar rats divided in 3 groups were administered sequentially with test item by oral gavage. The first set of 3 female rats was given a single dose of 300 mg/kg body weight. No mortality was observed at this dose level, so the second set of 3 female rats was treated at the same dose level. No mortality was observed at this dose level, so the third set of 3 female rats was treated at the higher dose level of 2000 mg/kg bw. All the rats were found dead at this dose level. Clinical sign like lethargy was observed from day 6 until day of death and also decrease in body weight was observed on day 7 in rats treated at this high dose. External examination of found dead and terminally sacrificed rats did not reveal any abnormality. Visceral examination of found dead rats revealed pale liver whereas terminally sacrificed rats did not reveal any lesion. Based on these results, the LD50 of the test item is lower than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off of the test item may be considered to be 500 mg/kg body weight by oral route in the rat.