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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 February 2020 - 26 February 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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

Constituent 1
Chemical structure
Reference substance name:
2-(ethylnitroamino)ethyl nitrate
EC Number:
285-332-6
EC Name:
2-(ethylnitroamino)ethyl nitrate
Cas Number:
85068-73-1
Molecular formula:
C4H9N3O5
IUPAC Name:
ethyl(nitro)[2-(nitrooxy)ethyl]amine
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: VELAZ PRAHA, Czech Republic
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: Range 178-214 g (9 females)
- Fasting period before study: yes
- Housing: The animals were housed in plastic cages suspended on stainless steel racks, up to 3 animals per cage in a room equipped with central air-conditioning.
- Diet (e.g. ad libitum): The laboratory food ssniff (Spezialdiäten GmbH, Germany) was offered at recommended doses each day approximately at the same time.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25ºC
- Humidity (%): 50-60%
- Air changes (per hr): Not specified.
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 60 mg/mL for the dose of 300 mg/kg bw. Dose of 2000 mg/kg bw was administered directly without vehicle.
- Amount of vehicle (if gavage): 5 mL/kg
- Justification for choice of vehicle: The test item is not soluble enough in water, therefore Olive oil was used as vehicle. Oil is a standard vehicle according to OECD TG 423.
- Lot/batch no.: L91087

MAXIMUM DOSE VOLUME APPLIED: 5 mL/kg bw for the dose of 300 mg/kg bw and 1.5 mL/kg bw for the dose of 2000 mg/kg bw.

DOSAGE PREPARATION: The required amount of the test item at dose of 2000 mg/kg bw was administered directly. Doses of 300 mg/kg bw were mixed with vehicle shortly before administration. Vehicle was used in this case due to low administration volume/body weight from the point of view of practical realization of small amount aplication. The leftovers of dose preparations were disposed of accordingly to valid standard operation procedures at laboratory facility.

CLASS METHOD
- Rationale for the selection of the starting dose: Available information indicated that the test item was likely to be non-toxic regarding acute toxicity. A limit dose of 2000 mg/kg body weight was therefore used as a starting dose.
Doses:
2000 and 300 mg/kg bw.
No. of animals per sex per dose:
3 female rats (2000 mg/kg) and 6 female rats (300 mg/kg)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed individually immediately after administration of the test item and 0.5, 1, 2, and 4 hours later. Each animal was inspected daily for the next 14 days. Individual weights of animals were measured immediately prior to test item administration and weekly thereafter.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: changes in skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity, and behavioural pattern. Particular attention was given to potential neurologic endpoints such as tremors, 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:
500 mg/kg bw
Based on:
test mat.
Mortality:
Mortality of 3/3 females at limit dose of 2000 mg/kg body weight.
Mortality of 1/6 females at dose of 300 mg/kg body weight.
At 2000 mg/kg the test item caused mortality of all animals within the day of administration. In a second step, 3 females were treated at dose of 300 mg/kg and no mortality was observed during 14-days observing period. In the next step the dose of 300 mg/kg caused death of one animal within the day of administration.
Clinical signs:
other: At the dose of 2000 mg/kg lethargy, vasodilatation, dyspnoea and spasms were observed in the animals before dying. In the second step, dose of 300 mg/kg did not cause signs of toxicity, change of health or any other adverse reactions. In the next step at
Gross pathology:
At the dose of 2000 mg/kg only autolytic changes were observed in 2 animals.
At the dose of 300 mg/kg the animal found dead had several 1-5 mm hemorrhages scattered areas in the lungs and rigor mortis (stiffening) occurred almost simultaneously with death. No macroscopic findings were observed in remaining animals at this dosage.

Any other information on results incl. tables

Table 1. Clinical Observations – 2000 mg/body weight, rats No. 1, 2, 3

Observation

Time After Administration

 Hour

Day

I

0.5

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Skin and Hair**

-

1,2,3

1,3

1,3

1,3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Eyes

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Mucosa

-

-

-

-

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Respiratory System*

2

-

1,3

1,3

1,3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Circulatory System

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

CNS

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Somatomotoric Activity

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Tremor

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Spasms

-

3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Salivation

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Diarrhoea

-

-

-

-

-

-

 -

-

-

-

-

-

-

-

-

-

-

-

-

Lethargy

1,2,3

1,3

1,3

1,3

1,3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sleep

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Coma

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Death

-

2

-

-

1,3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sacrificed

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Others

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-No observed signs, I- immediately, *-dyspnoe, **-vein vasodilatation in hindlimb soles and tail

Sex

Dose

ID

Administration

 Result

Clinical Observation

2000 mg/kg

1

death

·lethargy immediately and dyspnoe from the first hour after administration of the test item until death (within the day of the test item administration)

·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death

2

death

·lethargy and dyspnoe immediately after administration of the test item until death (within half an hour after administration of the test item)

·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death

3

death

·lethargy immediately and dyspnoe from the first hour after administration of the test item until death (within the day of the test item administration)

·spasms occurred within half an hour after administration of the test item and lasted 30 seconds in lateral recumbency

·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death

Table 2. Clinical Observations – 300 mg/body weight, rats No. 4, 5, 6

Observation

Time After Administration

 Hour

Day

I

0.5

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Skin and Hair

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Eyes

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Mucosa

-

-

-

-

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Respiratory System

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Circulatory System

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

CNS

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Somatomotoric Activity

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Tremor

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Spasms

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Salivation

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Diarrhoea

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Lethargy

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sleep

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Coma

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Death

-

2

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sacrificed

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Others

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-No observed signs, I- immediately.

Sex

Dose

ID

Administration

Result

Clinical Observation

300

mg/kg

4

alive

·no signs of intoxication, nor adverse reactions during the 14-day observation period

5

alive

·no signs of intoxication, nor adverse reactions during the 14-day observation period

6

alive

·no signs of intoxication, nor adverse reactions during the 14-day observation period

Table 3. Clinical Observations – 300 mg/body weight, rats No. 7, 8, 9

Observation

Time After Administration

 Hour

Day

I

0.5

1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Skin and Hair**

-

7,9

9

9

9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Eyes

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Mucosa

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Respiratory System*

7,8

8,9

8

8

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Circulatory System

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

CNS

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Somatomotoric Activity

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Tremor

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Spasms

7

9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Salivation

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Diarrhoea

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Lethargy

7,8

8,9

8,9

8,9

8,9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sleep

-

-

9

9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Coma

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Death

-

7

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Sacrificied

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Others

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-No observed signs, I- immediately, *-dyspnoe, **-vein vasodilatation in hindlimb soles and tail

Sex

Dose

ID

Administration

Result

Clinical Observation

300 mg/kg

7

death

·dyspnoe and lethargy immediately, spasms occurred 10 min after administration of the test item and lasted 20 seconds in lateral recumbency, death half an hour after administration

·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death

8

alive

·dyspnoe immediately and lasted until the 2nd hour after administration of the test item, lethargy immediately until the 4thhour after administration of the test item

9

alive

·dyspnoe after half an hour after administration of the test item, spams occurred half an hour after administration of the test item and lasted 30 seconds in lateral recumbency, lethargy half an hour until the 4thhour after administration of the test item, sleep after one hour until the 2ndhour after administration

·vein vasodilatation in hindlimb soles and tail occurred after half an hour until the 4thhour after administration

Table 4. Body weight

Sex

Dose

ID

Body Weight (g)

Body Weight Difference (g)

Initial

Week 1

Week 2

Week 1-Initial

Week 2-Initial

Week 2-Week 1

2000 mg/kg

1

205

-

-

-

-

-

2

207

-

-

-

-

-

3

214

-

-

-

-

-

300 mg/kg

4

188

224

240

36

52

16

5

200

235

255

35

55

20

6

178

226

242

48

64

16

300 mg/kg

7

195

-

-

-

-

-

8

207

240

246

33

39

6

9

202

242

252

40

50

10

Table 6. Necropsy results

Sex

Dose

ID

Result

2000 mg/kg

1

autolytic changes
(animal was found dead (after rigor mortis) and its body already started to self-digest/decompose.

In this state we cannot describe any changes made by the TI)

2

no visible pathological changes

3

autolytic changes

300 mg/kg

4

no visible pathological changes

5

no visible pathological changes

6

no visible pathological changes

300 mg/kg

7

The lungs scattered areas of several 1-5 mm hemorrhages

Rigor mortis (stiffening) occur almost simultaneously with death

8

no visible pathological changes

9

no visible pathological changes

Applicant's summary and conclusion

Interpretation of results:
other: Category 4 (CLP Regulation EC no. 1272/2008)
Conclusions:
The oral LD50 of the test item in female rats was found to be greater than 300 mg/kg bw and lower than 2000 mg/kg bw. The LD50 cut-off of the test item may be considered to be 500 mg/kg bw.

Executive summary:

The potential acute toxicity of the test item was studied on female Wistar rats, according to OECD TG 423, under GLP conditions. Since available information indicated that the test item was likely to be non-toxic regarding acute toxicity, a first step was performed by administering a single dose of 2000 mg/kg bw test item to three animals by gavage. The test item caused mortality of 3 animals within the day of administration of the test item. In a second step, 3 females were treated at the dose of 300 mg/kg bw. Test item-related mortality was not observed during the 14-days observing period. In the next step the same dose of 300 mg/kg caused death of one animal within the day of administration. At doses of both 2000 and 300 mg/kg lethargy, vasodilatation, dyspnoea and spasms were observed in the animals. The body weight of all surviving animals increased during the study. During necropsy, at the dose of 2000 mg/kg only autolytic changes were observed in 2 animals. At the dose of 300 mg/kg the animal found dead had several 1-5 mm hemorrhages scattered areas in the lungs and rigor mortis (stiffening) occurred almost simultaneously with death. No macroscopic findings were observed in remaining animals at this dosage. Based on the results, the LD50 of the test item is determined to be greater than 300 mg/kg bw and lower than 2000 mg/kg bw. Based on Annex 2d Test Procedure with a Starting Dose of 2000 mg/kg body weight of OECD Guideline 423, it can be concluded that the test item is classified in GHS Category 4 with a LD50 cut off value 500 mg/kg bw.

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