2-ethylhexyl nitrate

Endpoint summary

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No analytical certificate (no data on batch number and expiry date of the test substance), no data on GLP and experimental conditions. The very high tested dose enables a reliable conclusion.

Qualifier:

according to guideline

Guideline:

other: Federal Hazardous Substance Act.

GLP compliance:

no

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory Supply Company, Indianapolis, Indiana
- Weight at study initiation: 200 to 275 g

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

Doses:

10 mL/kg

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: no data
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs

Preliminary study:

Yes, but results were not indicated.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 10 mL/kg bw

Based on:

test mat.

Mortality:

2 males and 1 female were found dead.

Clinical signs:

other: No clinical signs were observed.

Gross pathology:

No gross abnormalities were noted at necrospy.

Interpretation of results:

other: inconclusive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The LD50 was expressed in mL/kg. The volume mass of the substance is 0.96 kg/L (0.96 g/mL). Therefore, when treating rats with 10 mL/kg, rats received 9.6 g/kg of substance, lowest limit of the LD50.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 9 600 mg/kg bw

Quality of whole database:

Only one study is available which has been given a reliability rating of 2, despite a number of limitations.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2020 to 15 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

yes

Remarks:

The mean relative humidity in the exposure chamber was below target range, but this was not considered to impact on interpretation of the study results / integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: VeryOne, Sample 2020
- Purity: >99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (approximately 23°C) and protected from light

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Spain S.L.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Housing: 3 animals per cage
- Diet: Global diet, ad libitum except during exposure
- Water: Tap water via bottles, ad libitum except during exposure
- Acclimation period: at least 5 days prior to exposure
- Microbiological status when known: Specific Pathogen Free (SPF)
- Method of randomisation in assigning animals to test and control groups: Animals randomly distributed by means of the body weight stratification method (during the acclimatization period) before the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-21.9°C
- Humidity (%): 35-58%
- Photoperiod: 12 hrs light / 12 hrs dark

IN-LIFE DATES: From: 09 June 2020 To: 23 June 2020

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

3.65 µm

Geometric standard deviation (GSD):

1.77

Remark on MMAD/GSD:

Mean Mass Median Aerodynamic Diameter (MMAD) of particle size distribution during exposure was calculated from two gravimetric measurements PSD #1 and PSD #2 (3.80 and 3.50 μm, respectively). Mean MMAD during exposure was 3.65 μm. This value was within the target range (1-4 μm).

Geometric Standard Deviations (GSD) on PSD #1 and PSD #2 were 1.76 and 1.78 respectively, and therefore two measurements were within the target range (1.5 - 3.0).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: EC-FPC-232 (anodized aluminium) equipped with glass exposure tubes
- Exposure chamber volume: approximately 3 litres
- Method of holding animals in test chamber: Animals were confined separately in restraint tubes which were positioned radially around the exposure chamber.
- Rate of air (airflow): 1.08 L/min (measured)
- System of generating aerosols: Liquid aerosol was generated from the liquid test item using a nebulizer (Hudson RCI, 41897, Side-draft NEB-U-MIST, Teleflex Inc.) The aerosol was diluted with filtered air from a compressor and conveyed via glass tubing from the generator to the exposure chamber. The flow rate through the exposure chamber was adjusted as necessary.
- Method of particle size determination: Determined gravimetrically twice during exposure, by analysing the test item deposited on each stage of a PIXE cascade impactor. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel.
- Temperature in air chamber: 19.2-20.7°C (mean: 19.73°C)
- Humidity in air chamber: 8.4-13.7% (mean: 10.35%)

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: Aerosol concentration was determined by trapping the test item in Acetone (Supelco (Merck), batch# I1063512 003) and analyzing the concentration in the trap by GC-FID (Agilent Technologies, GC 7890, Santa Clara, USA) according to a method previously validated at the testing facility. Optimum trapping conditions were determined during technical trials performed without animals before the in-life phase of the study; a sampling time of 5 minutes was chosen for the main study. During the exposure period, analytical determination of the aerosol concentration was performed eight times. Quality control samples for each step were analysed before the sample analysis to assure the correct concentrations of the test item.

VEHICLE
- Composition of vehicle: The aerosol was diluted with filtered air from a compressor and conveyed via glass tubing from the generator to the exposure chamber

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.65 µm (mean MMAD) / 1.77 (mean GSD)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5 mg/l (target); 5.65 mg/l (mean actual concentration)

No. of animals per sex per dose:

3/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
Mortality and morbidity examinations – daily
Clinical signs – hourly during exposure (only grossly abnormal signs), immediately after and 1 hour post-exposure, then once daily until the end of the observation period
Body weight – once during the acclimatization period, on the day of treatment just before the exposure, then on days 2, 4, 8 and day of gross necropsy

- Necropsy of survivors performed: yes. Sacrifice via an intraperitoneal overdose of pentobarbital.
- Other examinations performed: mortality, clinical signs, body weight, gross necropsy (examination of abdominal and thoracic cavities and contents; special attention given to any respiratory tract changes)

Statistics:

No statistical analysis was required

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.65 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All male and female animals lived through the 4-hour exposure period. However, one female died within 24 hours following the exposure to the test item.
No mortality was recorded for the rest of the animals and the remaining five animals completed the clinical observation period.

Clinical signs:

other: See "other findings" section for details

Body weight:

All animals lost weight between the exposure day and the 24 hours post-exposure. Following 24 hrs post-exposure, animals started to gain weight and this trend continued for the remaining observation period. The weight loss was attributed to the stress induced by the nose-only exposure protocol.

Gross pathology:

No macroscopic findings were observed in any of the animals during the necropsy.

Other findings:

All males (ID1, ID2 and ID3) showed loss of stability, piloerection, dirty fur (back), liquid secretion from nose, wet fur (abdomen and chest) and disorientation following test item exposure. Additionally, ID2 and ID3 had chromorrhinorrhea, chromodacryorrhea (in both eyes), ID1 had exophthalmos (both eyes), respiratory crackles and wet fur (head). ID2 and ID3 displayed respiratory crackles and excessive salivation respectively. Following 24 hours post-exposure, all clinical symptoms disappeared and animals did not display any clinical signs for the remaining of the observation period.

One female (ID4) died shortly after the exposure. The remaining two females (ID5 and ID6) showed loss of stability, piloerection, dirty fur (back), excessive salivation, liquid secretion from nose, wet fur (abdomen and chest) and disorientation. ID6 additionally had chromorrhinorrhea, chromodacryorrhea (in both eyes), exophthalmos (both eyes), breathing difficulty and wet fur (head). Following 48 hours post-exposure, all clinical symptoms disappeared and for the remainder of the observation period no clinical signs were observed.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

In this study, the LC50 is >5.65 mg/l. However, based on the death of one female out of the six animals exposed to the test concentration (5.65 mg/l), it is concluded that category 5 is appropriate according to GHS criteria (>5-12.5 mg/l for aersols) and OECD Test Guideline 436, but that no classification is required under CLP.

Executive summary:

The present study has been designed to evaluate the acute inhalation toxicity of the test item 2-ethylhexyl nitrate in male and female Sprague Dawley rats by the acute toxic class (ATC) method, OECD test guideline: Nº 436. This method allows the classification of the test item according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

For that purpose, a group of three male and three female Sprague Dawley rats was exposed by nose-only flow-past inhalation to the test item at a target concentration of 5mg/L for 4 hours.

All animals were observed for mortality and clinical signs during the exposure and the subsequent 14-day observation period until the day of gross necropsy. Body weights were recorded during acclimatization, on the day of treatment just before the exposure and on days 2, 4, 8 and on the day of necropsy.

All surviving animals were subjected to a gross necropsy and descriptions of all macroscopic abnormalities were recorded if any.

The ranges of aerosol concentrations, temperature, relative humidity and air flow rate were considered satisfactory for a study of this type. In addition, the test item was considered to be respirable by the rats. The mean analytical aerosol concentration was 5.65 mg/L. The deviation from the target aerosol concentration was 13 percent.

One female animal died within 24 hours following the exposure, but all other animals survived until gross necropsy at the end of the observation period. Animals displayed a number of clinical signs, including wet fur (abdomen, chest, back), chromorrhinorrhea, lachrymation and chromodacryorrhea in both eyes. All these clinical symptoms were no longer present at 48 hours post-exposure. For the rest of the observation period, animals did not show any abnormal clinical signs.

All animals lost weight between the exposure day and the 24 hours post-exposure. Following 24 hrs post-exposure, animals started to gain weight and this trend continued for the remaining observation period.

No macroscopic findings were observed in any of the animals during the necropsy.

Under the present experimental conditions, it can be concluded that the LC50 for the test item is >5.65 mg/l. However, based on the death of one female out of the six animals exposed to the test concentration, it is concluded that category 5 is appropriate according to GHS criteria (LC50 >5-12.5 mg/l for aerosols) and OECD Test Guideline 436, but that no classification is required under CLP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

> 5.65 mg/L air

Physical form:

inhalation: aerosol

Quality of whole database:

Only one study is available which has been given a reliability rating of 1, as it was conducted according to OECD Test Guidelines and GLP, with only one minor deviation.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Only 4 instead of 5 tested animals + unknown shaved area surface (cm2) + no mention of application under gauze + no detail on investigations (unknown whether any post-mortem examination done) + number of deaths not indicated = doubtful sensitivity and lack of details. The skin was abraded before treatment. However the moderate skin penetration (see 7.1) and the absence of acute oral toxicity imply an absence of dermal toxicity.

Qualifier:

according to guideline

Guideline:

other: Federal Hazardous Substance Act

GLP compliance:

no

Test type:

fixed dose procedure

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Small Stock Industries, Pea Ridge, Arkansas
- Age at study initiation: young adults
- Weight at study initiation: 2.5 to 3.5 kg

Type of coverage:

not specified

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: not indicated
- % coverage: not indicated
- Type of wrap if used: rubber dam

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with lukewarm water
- Time after start of exposure: just after removal of the rubber dams

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 mL/kg

Duration of exposure:

24 hours

Doses:

5 mL/kg

No. of animals per sex per dose:

4 animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: not indicated
- Necropsy of survivors performed: not indicated
- Other examinations performed: clinical signs

Preliminary study:

Not indicated

Sex:

not specified

Dose descriptor:

LDLo

Effect level:

> 5 mL/kg bw

Based on:

test mat.

Mortality:

None

Clinical signs:

other: None

Gross pathology:

Not indicated.

Interpretation of results:

other: inconclusive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The LDLo of the test substance after skin exposure during 24 hours is > 5 mL/kg. According to the volume mass of the test substance (0.96 kg/L), the LDLo of the test substance under the experimental conditions of the study is > 4.8 g/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD0

Value:

> 4 800 mg/kg bw

Quality of whole database:

Only one study is available which has been given a reliability rating of 2, despite a number of limitations.

Additional information

Animal data suggest a total absence of toxicity by oral and dermal routes. Data from the inhalation route indicate that classification in category 5 would be appropriate.

However, there are reports of effects in workers including dizziness, headaches. Organic nitrates have vasodilatation properties that could account for such effects. There is also a problem of habituation to their effects, followed by possible effects upon cessation of exposure.

Justification for classification or non-classification

The results from oral and dermal animal studies do not indicate that classification is required according to GHS and CLP criteria. The results from the acute inhalation animal study indicate that category 5 would be appropriate according to GHS criteria and OECD Test Guideline 436, but that no classification would be required under CLP. However, as a worst-case, Acute tox 4 (H302, H312, H332) is proposed for all routes of exposure based on human data (corresponding dose-levels unknown).