2-(methylnitroamino)ethyl nitrate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2020 - 28 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

Test material information

Constituent 1

Reference substance name:

2-(methylnitroamino)ethyl nitrate

EC Number:

241-168-7

EC Name:

2-(methylnitroamino)ethyl nitrate

Cas Number:

17096-47-8

Molecular formula:

C3H7N3O5

IUPAC Name:

methyl(nitro)[2-(nitrooxy)ethyl]amine

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: As the test item was phlegmatized in 15 % water for safety reasons during transportation, before the test item preparation for administration, the test item was gentle dried in temperature 20 ± 5 ºC during 24 hours.

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 192-212 g (12 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 (dose of 300 mg/kg) and 400 mg/mL (dose of 2000 mg/kg)
- 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

DOSAGE PREPARATION: The required amount of the test item (according to the body weight) was mixed with vehicle (olive oil) shortly before administration. Administration volume was 5 mL/kg. 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:

6 female rats.

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 levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - <= 2 000 mg/kg bw

Based on:

test mat.

Effect levels 2

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

The test item in step 1 at dose of 2000 mg/kg bw caused mortality of one animal within half an hour after administration. In the second step at 2000 mg/kg bw, test item-related mortality was observed in all 3 animals within 4 hours after administration. The test item in the next step the dose of 300 mg/kg caused death of one animal immediately after administration. The last step in a dose of 300 mg/kg did not cause any death. In conclusion:
Mortality of 4/6 females at a limit dose of 2000 mg/kg body weight.
Mortality of 1/6 females at a dose of 300 mg/kg body weight.

Clinical signs:

other: Rats displayed signs such as: dyspnoea, vasodilatation, spasms and lethargy. Summary results of clinical observations are presented in tables 1, 2, 3, 4.

Gross pathology:

At the dose of 2000 mg/kg bw, the necropsy showed scattered hemorrhages areas in lungs and early onset of the rigor mortis in the four died animals. No visible pathological findings after 14-days period occurred in the remaining animals. At 300 mg/kg bw dose, the necropsy of the only died animal showed hemorrhage of the lungs and early onset of rigor mortis. The macroscopic examination of remaining animals dosed with 300 mg/kg bw of test item did not reveal any pathological changes.

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**	2,3	3	3	3	3	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Eyes	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mucosa	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Respiratory System*	2,3	2,3	1,3	3	3	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Circulatory System	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
CNS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Somatomotoric Activity	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Tremor	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Spasms	2	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Salivation	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Diarrhoea	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Lethargy	2,3	2,3	3	3	3	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sleep	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Coma	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Death	-	2	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
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	alive	· Dyspnoe occurred one hour after administration of the test item
		2	death	·lethargy and dyspnoe immediately after administration of the test item until death (within half an hour after administration of the test item) ·spasms occurred immediately after administration of the test item and lasted 30 seconds in lateral recumbency ·vein vasodilatation in hindlimb soles and tail occurred immediately until death
		3	alive	·lethargy and dyspnoe immediately after administration of the test item until 4^thhour ·vein vasodilatation in hindlimb soles and tail occurred immediately until until 4^thhour

Table 2. Clinical Observations – 2000 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**	-	4,5,6	4,5,6	6	6	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Eyes	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mucosa	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Respiratory System*	5	4,5,6	6	6	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Circulatory System	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
CNS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Somatomotoric Activity	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Tremor	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Spasms	5		-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Salivation	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Diarrhoea	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Lethargy	-	4,5	6	6	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sleep	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Coma	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Death	-	-	4,5	-	6	-	-	-	-	-	-	-	-	-	-	-	-	-	-
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	4	death	·dyspnoe and lethargy half an hour after administration of the test item until death (within an hour after administration of the test item) ·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death
		5	death	·dyspnoe immediately and lethargy half an hour after administration of the test item until death (within an hour after administration of the test item) ·spasms occurred immediately after administration of the test item and lasted 20 seconds in lateral recumbency ·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death
		6	death	· dyspnoe after half an hour and lethargy in hour after administration of the test item until death (within 4 hours after administration of the test item) ·vein vasodilatation in hindlimb soles and tail occurred after half an hour until death

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**	-	-	-	8,9	8,9	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Eyes	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mucosa	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Respiratory System*	7,8,9	8,9	8,9	8,9	8,9	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Circulatory System	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
CNS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Somatomotoric Activity	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Tremor	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Spasms	7,9	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Salivation	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Diarrhoea	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Lethargy	9	9	9	9	9	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sleep	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
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 immediately, spasms occurred 10 min after administration of the test item and lasted 20 seconds in lateral recumbency, death half an hour after administration
		8	alive	·dyspnoe immediately and lasted until the 4^thhour after administration ·vein vasodilatation in hindlimb soles and tail occurred after two hours until the 4^thhour after administration
		9	alive	·dyspnoe and lethargy immediately after administration of the test item until the 4^thhour after administration of the test item, spams occurred 10 min after administration of the test item and lasted 30 seconds in lateral recumbency ·vein vasodilatation in hindlimb soles and tail occurred after two hours until the 4^thhour after administration

Table 4. Clinical Observations – 300 mg/body weight, rats No. 10, 11, 12

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**	-	-	-	10	10	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mucosa	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Respiratory System*	10,11,12	10, 11, 12	10, 11, 12	10, 11, 12	10, 11, 12	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Circulatory System	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
CNS	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Somatomotoric Activity	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Tremor	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Spasms	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Salivation	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Diarrhoea	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Lethargy	10,11,12	10, 11, 12	10, 11, 12	10, 11, 12	10, 11, 12	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sleep	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Coma	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Death	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Sacrificied	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Others	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

-No observed signs, I- immediately, *-dyspnoe, **-orbital tightening

Sex	Dose	ID	Administration Result	Clinical Observation
♀	300 mg/kg	10	alive	·dyspnoe and lethargy immediately until the 4^thhour after administration of the test item ·orbital tightening after two hours until the 4^thhour after administration of the test item
		11	alive	·dyspnoe and lethargy immediately until the 4^thhour after administration of the test item
		12	alive	·dyspnoe and lethargy immediately until the 4^thhour after administration of the test item

Table 5. 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	209	250	268	41	59	18
		2	202	-	-	-	-	-
		3	193	224	247	31	54	23
		4	206	-	-	-	-	-
		5	212	-	-	-	-	-
		6	210	-	-	-	-	-
	300 mg/kg	7	200	-	-	-	-	-
		8	210	245	251	35	41	6
		9	201	242	248	41	47	6
		10	192	220	236	28	44	16
		11	210	233	242	23	32	9
		12	198	222	234	24	36	12

Table 6. Necropsy results

Sex	Dose	ID	Result
♀	2000 mg/kg	1	no visible pathological changes
		2	The lungs scattered areas of several 1-5 mm hemorrhages Rigor mortis (stiffening) occur almost simultaneously with death
		3	no visible pathological changes
		4	The lungs scattered areas of several 1-5 mm hemorrhages Rigor mortis (stiffening) occur almost simultaneously with death
		5	The lungs scattered areas of several 1-5 mm hemorrhages Rigor mortis (stiffening) occur almost simultaneously with death
		6	The lungs are diffusely dark red Rigor mortis (stiffening) occur almost simultaneously with death
	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
		10	no visible pathological changes
		11	no visible pathological changes
		12	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 2000 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 one animal within half an hour after administration of the test item. In a second step, 3 females were treated at the same dose of 2000 mg/kg bw. Test item-related mortality was observed in all 3 animals within four hours after administration. The test item in the next step of a dose 300 mg/kg bw caused death of one animal immediately after administration. At the dose of 2000 mg/kg lethargy, vasodilatation, dyspnoea and spasms were observed in all animals. During the necropsy, scattered hemorrhages areas in lungs and early onset of the rigor mortis were observed in died animals. Surviving animals displayed no visible pathological changes. The dose of 300 mg/kg administered caused signs of lethargy, vasodilatation, dyspnoea, and spasms in all animals. The macroscopic examination of animals dosed with 300 mg/kg did not reveal any pathological changes except for the dead animal which showed scattered lung hemorrhages and early onset of the rigor mortis. The body weight of all surviving animals increased during the study. 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 2000 mg/kg bw.