2-ethylhexyl nitrate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed with the rapidly formed hydrolysis product of the target chemical following internationally validated test guidelines and GLP requirements, reported in sufficient detail; publication meets generally accepted standards

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

1981

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Ethylhexanol

IUPAC Name:

2-Ethylhexanol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: supplied by Dr. K. Thomae GmbH, D-88400 Biberach/Riss, Germany
- Age at study initiation: About 7 weeks old on delivery
- Weight at study initiation: Mean body weight males ca. 238 g (standard error of the mean max. ± 2.3 g), females 170 g (standard error of the mean max.± max. 2.2 g)
- Housing: The test animals were individually kept in wire cages placed in the inhalation chambers
- Diet: KLIBA rat/mouse laboratory diet 24-343-4 (Klingentalmühle, AG, CH-4303 Kaiseraugst, Switzerland) ad libitum in the exposure-free periods
- Water: Ad libitum in the exposure-free periods
- Acclimation period: 5 days before exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: Day 1 To: Day 93

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Remarks:

inhalation chamber

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Horizontal-flow whole-body exposure system, chamber volume ca. 1.1 m³ (manufactured using glass & steel by BASF AG, Ludwigshafen, Germany)
- Method of holding animals in test chamber: Individually in wire cages
- Source: Ambient air for 15 ppm exposure, compressed air for 40 and 120 ppm concentrations, exchange rate not stated
- Method of conditioning air: Warmed air of the control group (45.7 °C) and the vapour/air mixture of the treatments were mixed with the overall stream generated by underpressure
- Temperature, humidity, pressure in air chamber: 21.3 to 23.8 °C (measured continuously), 41.8 to 46.2 % (recorded once daily), overpressure 10.1 Pa in the treatments and -10.2 underpressure in the controls

TEST ATMOSPHERE
- Brief description of analytical method used: GC-FID
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the inhalation atmospheres were analysed at intervals of about 15 min by gas chromatography (Hewlett Packard gas chromatograph Model HP 5880 A with automatic sampler HP 7671 A, FID, column: 1m ∙ 2 mm with 10 % Triton x 305 on Supelcoport, 102/120 mesh, oven temperature: 120°C. C15-parafin used as the internal standard).

Duration of treatment / exposure:

90 days (sacrifice on Day 94)

Frequency of treatment:

6 h per day, 5 days per week (65 exposures)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 15, 40 and 120 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 15.0±0.6, 39.9±1.33 and 120±4.8 [ppm±SD]
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest test concentration of 120 ppm corresponds to the calculated saturated vapour concentration at 20 °C. Higher concentrations obtainable as aerosols were considered irrelevent for occupational exposure. The remaining concentrations were included to potentially establish a concentration-effect function.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, clinical signs and findings were recorded
- Time schedule: During exposure and daily at exposure free times

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before start and after end of exposure

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were determined weekly from test start to end, additionally at the beginning of the pre-flow period (i.e. 5 days before test start) and 1 day before start of exposure

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to beginning of the preflow-period and after termination
- Dose groups that were examined: All treatments.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from all animals at the end of the 3–month exposure period, Day 94
- Parameters examined: White blood cells, red blood cells, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, differential blood count and clotting time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from all animals at the end of the 3–month exposure period. Day 94
- Animals fasted: No
- Parameters examined: sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and cyanide-insensitive palmitoyl-CoA oxidation, a marker for peroxisome proliferation

Sacrifice and pathology:

Sacrifice at Day 94
GROSS PATHOLOGY: complete necropsy of all animals including weighing of certain organs, including lungs, liver, kidneys, adrenal glands and testes and a gross–pathologic evaluation was performed.
HISTOPATHOLOGY: selected organs/tissues were examined histologically based on OECD testing guideline

Statistics:

Mean values and standard deviation calculated for body weight, body weight change, haematological and clinical biochemistry parameters, absolute and relative organ weights.
Organ weights: Dunnett's test for comparison of treatments with control.
Analysis of variance with subsequent Dunnett's test to compare body weight, body weight change and haematological & clinical biochemistry of treatments with control

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain of female animals of 40 and 120 ppm decreased compared to controls on day 37. Males of the 15 ppm group had a statistically higher increase in bodyweight on day 93 compared to controls. Differences are not treatment related (table 1.)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Differences observed were: (1) 120 ppm group, males: bilirubin = 4.07 mmol/L (controls 2.99 mmol/L); (2) 15 ppm group females: glucose = 6.98 mmol/L (controls 7.81 mmol/L)

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

Discriminating level = highest test concentration, 90 days, 6 h per day on 5 days per week, 65 exposures

Effect level:

>= 120 ppm (analytical)

Based on:

test mat.

Remarks:

2-Ethylhexan-1-ol, measured concentration, SD ±4.8 ppm

Sex:

male/female

Basis for effect level:

other: Absence of toxicity at highest achievable vapour concentration with the experimental setting applied (vapour saturation at 20°C),

2

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 639 mg/m³ air

Based on:

test mat.

Remarks:

2-Ethylhexan-1-ol

Sex:

male/female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 863 mg/m³ air

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate, equimolar

Sex:

male/female

Basis for effect level:

other: see 'Remark'

4

Dose descriptor:

NOAEC

Remarks:

Discriminating level

Effect level:

>= 5 other: mmol/m³ air

Based on:

other: molar concentration of source and target chemical

Sex:

male/female

Basis for effect level:

other: no toxicity at highest achievable vapor

5

Dose descriptor:

NOAEL

Remarks:

Discriminating dose

Effect level:

>= 125 other: mg/kg bw/day (inhalation-to-oral extrapolation)

Based on:

other: conversion to target chemical 2-Ethylhexyl Nitrate, equimolar

Sex:

male/female

Basis for effect level:

other: With respiratory rate (6 h, rat) 0.29 m³/kg bw (ECHA Guidance Table R. 8-2, 2012), the corresponding dose-level is 863∙0.29∙50 %/100 %=125 mg/kg/day and considering 50 % inhalation and 100 % oral absorption.

6

Dose descriptor:

NOAEL

Remarks:

Discriminating level

Effect level:

>= 0.71 other: mmol/kg bw/day (inhalation-to-oral extrapolation)

Based on:

other: molar concentration of source and target chemical

Sex:

male/female

Basis for effect level:

other: no toxicity at highest achievable vapor

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Body weight measurements

Weighing time point:		Day 1	Day 9	Day 23	Day 37	Day 51	Day 65	Day 79	Day 93
Males/group
0 ppm	m	282.6	326.3	375.2	413.9	443.3	470.1	487.0	501.6
	sd	10.0	19.9	22.0	25.0	25.8	25.9	26.2	29.1
15 ppm	m	287.2	338.7	383.0	426.2	461.8	487.8	512.4	535.5*
	sd	11.6	11.9	13.8	19.1	18.9	20.6	27.5	32.5
40 ppm	m	286.4	333.7	375.6	421.2	452.1	479.1	499.4	520.0
	sd	8.3	14.8	20.3	24.6	33.3	34.5	40.8	37.7
120 ppm	m	285.5	327.3	372.1	409.2	438.9	463.6	483.3	497.0
	sd	7.3	11.0	13.2	15.3	17.6	18.4	21.4	19.4
Females/group
0 ppm	m	192.2	211.8	225.9	248.0	258.6	266.6	274.3	281.3
	sd	9.3	8.4	15.1	14.1	19.0	17.1	22.0	19.1
15 ppm	m	193.9	206.5	224.2	241.5	257.1	264.2	271.2	275.4
	sd	10.1	18.3	16.0	19.4	16.4	20.0	18.2	19.1
40 ppm	m	196.3	212.1	228.6	237.3	257.5	261.1	272.4	275.3
	sd	3.7	6.9	12.3	11.7	14.1	16.1	15.3	15.4
120 ppm	m	195.3	209.7	224.6	234.7	252.8	257.6	265.0	271.5
	sd	4.6	5.7	7.1	7.3	8.8	11.0	11.9	12.1

Statistically significant: *P < 0.05

m = mean value

sd = standard deviation

Applicant's summary and conclusion

Conclusions:

No substance-related adverse effects of the test item and source substance observed at the highest vapour concentration achievable, thus NOAEL ≥ 120 ppm (discriminating dose) for male and female rats; considering the significantly lower vapour pressure of the target chemical (27 versus 93 Pa at 20 °C) an equimolar exposure exceeding saturated vapour concentrations of the target chemical 2-Ethylhexyl Nitrate is concluded to be non-toxic.

Executive summary:

The subchronic repeated dose toxicity of the test item and source substance 2-Ethylhexan-1-ol (CAS 104-76-7) via the inhalation route in the rat was measured in a GLP-compliant study using the “Subchronic Inhalation Toxicity: 90-day Study” protocol compliant with OECD TG 413 (1981). The validity criteria were met and the experiment can be considered relevant and adequate for the endpoint. Therefore it is deemed conclusive and was rated „reliable without restrictions“, i.e. “Klimisch 1” according to the scale of Klimisch et al. (1997). The rating of the study for use in an analogue approach is lower compared to equally rated studies on the target substance.

Ten male and female Wistar rats were individually held in inhalation chambers for 90 days (6 hours perday, 5 days a week) and exposed to vapours of the test item via whole body exposure. While the treated chambers were maintained under ca. 10 Pa underpressure to ensure in flow of the prepared air/vapour mixtures, the control chambers had constant overpressure of 10 Pa from a clean air supply to prevent cross contamination. The exposure level were nominal 0, 15, 40 and 120 ppm and analytically determined employing GC-FID to 0, 15.0±0.6, 39.9±1.33 and 120±4.8 [ppm ± SD], which shows good agreement. The highest achievable test concentration of 120 ppm corresponds to the calculated saturated vapour concentration at 20 °C. Higher concentrations obtainable as aerosols were refused because of their considered irrelevance for occupational areas. The examinations performed included cage side and detailed clinical observations, body weights, ophthalmoscopic examination, haematology clinical chemistry, gross pathology and histology.

None of the followed parameters showed any treatment related change in either sex compared with the controls. Differences to the control group were observed in body weight gain and clinical chemistry. In the 40 and 120 ppm treatments the body weight gain of female test animals was decreased compared to the control on Day 37 only. The male rats in the 15 ppm treatment group showed statistically significantly higher increase in body weight on day 93 compared to control. Furthermore 120 ppm group male rats showed bilirubin f 4.07 mmol/L (controls 2.99 mmol/L) and female rats in the 15 ppm group had a glucose level of 6.98 mmol/L (controls 7.81 mmol/L). It was concluded that these differences were not of toxicological significance and were not treatment related.

In conclusion no substance-related adverse effects of the test item were observed at the highest vapour concentration achievable, which is thus considered ≥ NOAEL (discriminating dose) for male and female rats. This level corresponds to 639 mg test item/m³ air or 5 mmol/m³ as a basis for equimolar comparison in read-across approaches.

Reading-across to the target substance 2-Ethylhexyl nitrate (2 -EHN, CAS 27247-96-7) delivers on the same molar concentration an air level of 863 mg/m³ air. Taking the significantly lower vapour pressure of the target chemical (only 27 versus 93 Pa of 2 -EH at 20 °C) into account, an equimolar exposure exceeding saturated vapour concentrations of the target chemical can be concluded to be non-toxic.

• Klimisch HJ, Andreae M, Tillmann U (1997). A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data. DOI 10.1006/rtph.1996.1076 PMID 9056496 Regul Toxicol Pharmacol 25:1-5.