2-(ethylnitroamino)ethyl nitrate

Endpoint summary

Description of key information

Acute oral toxicity: Key study. Test method according to OECD 423, GLP study. 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.

Acute dermal toxicity: Key study. Test method similar to OECD 402, GLP study. The LD50 of the test item is higher than 2000 mg/kg body weight by dermal route in rabbits.

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:

04 February 2020 - 26 February 2020

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)

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: 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.

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.

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.

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 4^thhour 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 4^thhour after administration of the test item, sleep after one hour until the 2^ndhour after administration ·vein vasodilatation in hindlimb soles and tail occurred after half an hour until the 4^thhour 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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

Key study with Klimisch score = 1

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 1991 - 29 November 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

Species tested was different from the preferred one and the number of animals used was higher than that specified in the Guideline.

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CAMM Research Lab Animals, Wayne, NJ; Hazleton Research Products, Denver, PA; Buckshire Corporation, Perkasie, PA.
- Females nulliparous and non-pregnant: Not specified.
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: Male: 2051.6 g (SD = 221.97 g); Female: 2135.0 g (SD = 266.17 g)
- Fasting period before study: not specified.
- Housing: Rabbits were housed individually in cages sized in accordance with the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, National Research Council. Waste material was removed twice weekly. Cages and feeders were sanitized every two weeks.
- Diet (e.g. ad libitum): Ad libitum. Purina Lab Rabbit Chow H.FR. Food was checked daily and added or replaced as needed. Feeders are designed to reduce soiling, bridging and scattering.
- Water (e.g. ad libitum): Ad libitum. Drinking water (Fresh tap-water). Water was monitored for contaminants at periodic intervals according to Standard Operating Procedure PH-018.
- Acclimation period: the animals were acclimatized for at least 5 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC (±3ºC)
- Humidity (%): 30 to 70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: Not specified.
- Type of wrap if used: A square gauze patch was placed on the animals to cover the dosed area. The animals were wrapped with rubber dam and an elastic bandage to retard evaporation.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the skin sites were wiped with water and gauze.
- Time after start of exposure: 24h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg body weight
- Concentration (if solution): N/A
- Constant volume or concentration used: yes
- For solids, paste formed: N/A

Duration of exposure:

24 h

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations were recorded daily through Day 14. Body weights were recorded at initiation and on Days 7 and 14.
- Necropsy of survivors performed: yes. At termination, gross pathological findings were recorded and reported.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality ocurred during the study.

Clinical signs:

other: No clinical signs were observed in any animal receiving the test chemical.

Gross pathology:

No visible lesions were observed in any animal at terminal necropsy.

Table 1. Summary of Clinical Observations. N-Ethyl-2 Nitratoethyl Nitramine

Clinical signs	Sex	Hours	Days
		24	2	3	4	5	6	7	8	9	10	11	12	13	14
No signs	M	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	F	5	5	5	5	5	5	5	5	5	5	5	5	5	5

Table 2. Summary of mortality. N-Ethyl-2 Nitratoethyl Nitramine

Dose (mg/kg)	Sex	Nº of rabbits	Days													Total mortality
			2	3	4	5	6	7	8	9	10	11	12	13	14
2000	M	5	0	0	0	0	0	0	0	0	0	0	0	0	0	0/5
2000	F	5	0	0	0	0	0	0	0	0	0	0	0	0	0	0/5

Table 3. Summary of Body Weights (g). N-Ethyl-2 Nitratoethyl Nitramine

Animal Number	Sex	Initial	Day 7	Final
5271	M	2034	2186	2327
5272	M	2138	2192	2309
5273	M	2361	2521	2681
5274	M	1911	2137	2278
5275	M	1814	1934	2095
x		2051.6	2194.0	2338.0
S.D.		211.97	210.86	212.87
N		5	5	5
5276	F	2340	2503	2613
5277	F	1866	1990	2084
5278	F	2444	2636	2755
5279	F	1865	1984	2115
5280	F	2160	2284	2396
x		2135.0	2279.4	2392.6
S.D.		266.17	295.04	296.74
N		5	5	5

Table 4. Necropsy Observations (Incidence Values). N-Ethyl-2 Nitratoethyl Nitramine

Observation	Interim Death Incidence		Terminal Necropsy Incidence
No visible lesions	M	F	M	F
	-	-	5	5

Interpretation of results:

other: No category (CLP Regulation EC no. 1272/2008)

Conclusions:

The LD50 of the test item is higher than 2000 mg/kg body weight by dermal route in rabbits.

Executive summary:

In a dermal limit test (GLP study), one group of 10 New Zealand White rabbits (five males and five females) was exposed to the test substance at 2000 mg/kg for an exposure period of 24 h. Animals were observed for clinical signs and mortality once daily for fourteen days. No mortality and no clinical signs were observed in any animal receiving the test chemical. There were no apparent effects on mean body weight throughout the study. No visible lesions were observed in any animal at terminal necropsy. Based upon these observations, the estimated acute dermal LD50 (combined sexes) was determined to be greater than 2000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Key study with Klimisch score = 1

Justification for classification or non-classification

Based on the available data, the substance is classified for oral acute toxicity (category 4) according to CLP Regulation (EC) no. 1272/2008.