2-ethylhexyl nitrate

Acute Toxicity: inhalation

Administrative data

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

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 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

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl nitrate

EC Number:

248-363-6

EC Name:

2-ethylhexyl nitrate

Cas Number:

27247-96-7

Molecular formula:

C8H17NO3

IUPAC Name:

2-ethylhexyl nitrate

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

Test animals

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

Administration / exposure

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

Results and discussion

Effect levels

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.

Applicant's summary and conclusion

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.