2-ethylhexanal

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline 412 under GLP conditions. No marked deviations from the the OECD protocol.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Reference 2

Reference Type:

publication

Title:

Toxikologische Bewertung 2-Ethylhexanal Nr. 113

Author:

BG Chemie

Year:

2005

Bibliographic source:

ISSN 0937-4248 / www.bgchemie.de/toxikologischebewertungen

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexanal

EC Number:

204-596-5

EC Name:

2-ethylhexanal

Cas Number:

123-05-7

Molecular formula:

C8H16O

IUPAC Name:

2-ethylhexanal

Details on test material:

- Name of test material (as cited in study report): 2-ethylhexanal
- Physical state: liquid
- Analytical purity: >98 %
- Impurities (identity and concentrations): n-butanal 2.9-3.5% in the 250 ppm dose; n-butanal was stripped from test atmosphere using nitrogen and dissappeared within one day under detection limit (<0.2%) before testing
- Lot/batch No.: Aldrich Chemical Co., Gillingham, Dorset, UK; internal reference number: 95-0078
- Stability under test conditions: not reported
- Storage condition of test material: substance stored in brown glass bottles at 4 °C in the dark

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 39-45 days
- Weight at study initiation: weight control at day -8, -5, -3, replacement of animals with spares if weight variation ≥20 %
- Fasting period before study: no
- Housing: 5m and 5f per cage (mesh-wired cages, stainless steel)
- Diet: ad libitum, exept during exposure and laboratory investigations; animal food source: Mouse No.1 modified diet (R/M1(E) SQC.FG by SDS Special Diets Services, Witham, Essex, UK and BIBRA)
- Water: ad libitum, exept during exposure and laboratory investigations; water quality guidelines according to annex2 80/778/EEC
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5- 24.5
- Humidity (%): 46 -74
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 h (19.30-07.30 local time) /12 h (07.30-19.30 h local time)

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

nose/head only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: vapour was tested

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: head/nose-only inhalation chambers
- Method of holding animals in test chamber: individual housing in restraining tubes during exposure
- System of generating aerosols: test atmosphere was generated through passing (massflow controller) small, wetered amounts of nitrogen (0.3-2.3L/min) through separarte evaporation flasks containing 2-ethylhexanal; flask were kept in water bath (21.5-22.4 °C); generation in a ventilated cabinet; a material/nitrogen mixture was mixed with small amount of oxygen and pressurized air, which had been passed through wash bottle containing water for humidification (temperature-controlled water bath 27.0-28.8 °C)
- Temperature, humidity, pressure in air chamber: 20.4-21.3 °C, 54-56 %, air pressure not reported
- Air flow rate: pressurized air (ca. 26.0 L/min) and oxygen (ca. 0.2-0.5 L/min); 26.0 L/min for air/positive control, 26.3/27.2/28.6 L/min for low/mid/high concentrations
- Air change rate: not reported
- Oxygen concentration: checked daily because nitrogen was used for test atmosphere, 20.3-22.7 %

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromtaographic analysis (Vega, Carlo Erba, Italy)
- Samples taken from breathing zone: yes (with automatically switched gas sampling ports at regular intervals)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- concentration in each exposure was monitored approximately four times per hour
- Analytical mean actual concentrations (and deviations): 25.5 (0.4) ppm, 102.2 (2.7) ppm, 250.7 (2.8) ppm; corresponding to 0.14, 0.54 and 1.34 mg/L, respectively

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/d, 28 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.00; 0.12; 0.47; 1.04 mg/l (0, 25, 100, 250 ppm)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.00; 0.14; 0.54; 1.34 mg/l (0, 25.5, 102.2, 250.7 ppm)
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

other: negative control: air... (see attached file)

Details on study design:

Dose selection rationale: 250 ppm likely to result in toxic effects, 100 ppm likely to result in less severe effects than 250 ppm dose, 25 ppm expected no-effect level

Positive control:

- Name of positive control: 2-(ethylhexyl)phtalate (DEHP) as a positive control for peroxisome proliferating potential
- Positive control group: 5 animals (m+f)
- Administration of DEHP: through diet
- Supplier of positive control: Aldrich Chemical Co, Gillingham, Dorset, UK
- Physical state: clear, colourless liquid
- Purity of positive control: > 99.0 %
- Storage conditions: original container at RT

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice during exposure, check in the afternoon (shortly after exposure)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice during exposure, check in the afternoon (shortly after exposure)

BODY WEIGHT: Yes
- Time schedule for examinations: body weight records, 1. on animal receipt (day -8), 2. day -5 and -3 before study, 3. day 0, 4. twice weekly and 5. at necropsy
- weekly body weight gain was calculated

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Other: Food intake was measured twice weekly per cage ver 3-4 day intervals, calculation as gram weight gain per gram food consumed

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: No
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy from abdominal aorta of unfasted rats under diehtylether anaesthesia
- Animals fasted: No
- How many animals: all
- Parameters checked in table [No.1 Clinical parameters] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Other examinations:

- Histological examination: samples from liver of a few appointed rats (No. 2, 4, 11, and 13 air control; no. 32, 34 ,31 and 33 in 2-ethylhexanal group (250 ppm); no. 22, 24 , 1 and 3 positive control group) were taken for transmission electron microscopy examination (see table 2 for details on animals)
- Liver homogenate was retained for biochemical analysis of cyanide-insensitive pamitoyl-CoA oxidation + protein conc.
- Microsomes were prepared from liver homogenate: rate of lauric acid 11- and 12-hydroxylase + protein conc.

Statistics:

- Body weights and body weight gain, organ weight and haematology data: Dunett´s multiple comparison test
- Percetages of blood cells: Kruskal-Wallis non-parametric analysis of variance folloewd by Mann/Whitney U-test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities and no substance related effects were observed. Transient porphyrine accumulation in 3/10 animals and decreaseed grooming were observed at 250 ppm on some days during the first week of exposure.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decrease in body weight in males (250 ppm)

FOOD CONSUMPTION & FOOD EFFICIENCY
Minimal increase in food intake and minmal reduced food conversion efficiency relative to air controls

HAEMATOLOGY
Decrease of lymphocytes and an increase in neutrophils in m/f at 250 ppm, statistically significant only in f rats.

CLINICAL CHEMISTRY
- decreased cholesterol levels at 250 ppm 2-ethylhexanal but their triglyceride level was increased
- decreased percentage of lymphocytes and increased percentage of neutrophils in blood
- increases in alkaline phosphatase activity (m/f) at levels of 25 and 100 ppm

ORGAN WEIGHTS
- Dose dependent adrenal weight increase in f, in m only at 250 ppm
- Thymus weight decrease in m/f at 250 ppm
- Lung weight increase in m/f at 250 ppm

GROSS PATHOLOGY
No substance related effects observed

OTHER FINDINGS
Biochemistry studies:
- Protein content: no substance-related effect o whole or microsomal protein content; [DEHP positive control: increase in whole protein 108-109 % and microsomal protein 122 % of control]
- Cyanide-insensitive pamitoyl-CoA oxidation from whole homogenate (m/f) of control: 158 % m (250 ppm)/ 118-149 % f (100-250 ppm); [DEHP positive control: 1116 % m/944 % f]
- Microsomal lauric acid 11-hydroxylase activity (m/f) of control: 131 % m (250 ppm)/ 129-138 % f (100-250 ppm); [DEHP positive control: 463 % m/ 305 % f]
- Microsomal lauric acid 12-hydroxylase activity (m/f) of control: 139, 141 and 224 % m (25, 100 and 250 ppm)/ 128-181 % f (100-250 ppm); [DEHP positive control: 1173 % m/ 797 % f]

Histopathological examination:
No substance-related effects observed for 2-ethylhexanal (results for positive control DEHP, check final report)

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

0.54 mg/L air (analytical)

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency and compound intake; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

102.2 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; food efficiency and compound intake; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

3

Dose descriptor:

NOEL

Remarks:

peroxisome proliferation

Effect level:

25.5 ppm (analytical)

Sex:

male/female

4

Dose descriptor:

NOEL

Remarks:

peroxisome proliferation

Effect level:

0.14 mg/L air (analytical)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Peroxisome proliferating potential of 2-ethylhexanal (BIBRA study report, taken from BG Chemie publication, see data source for detailed information)

	Negative control	25 ppm	100 ppm	250 ppm	DEHP (1.2 % diet), positive control
Male rats - effects
Absolute liver weight (g)	6.47	6.64 ns	6.69 ns	6.71 ns	12.34 ^2
Relative liver weight (g/kg bw)	31.2	33.8 ^1	31.7 ns	35.4^2	59.0 ^2
Liver histopathology	NSRE	NSRE	NSRE	NSRE	Hepatocyte hypertrophy, marked cytoplamic eosinophilia, cellular necrosis 1/5 animals
Electron microscopy  of liver (number and morphology of peroxisomes an, and amount on smooth ER and in centrilobular and periportal hepatocytes )	NSRE	Not examined	Not examined	NSRE	Amount and size of peroxisomes marked increased peripotal and centrilobular, amount of smooth ER marked increase periportal and a week increase centrilobular
Total protein content in liver homogenat (mg/g liver)	202	200 ns	199 ns	205 ns	220 ^2
Microsomal protein content (mg/g liver)	33.4	33.4 ns	36.8 ^1	31.5	40.8 ^2
Cyanide-insensitive palmitoyl-CoA oxidase activity in whole liver homogenate (nmol/min/mg protein)	5.26	5.33 ns	5.62 ns	8.29 ^3	58.7 ^3
Lauric acid 11-hydroxylase activity in microsomal liver fraction (nmol/min/mg protein)	0.35	0.39 ns	0.39 ns	0.46 ^2	1.62 ^3
Lauric acid 12-hydroxylase activity in microsomal liver fraction (nmol/min/mg protein)	0.59	0.82 ^3	0.83 ^3	1.32 ^3	6.92 ^3
Female rats - effects
Absolute liver weight (g)	4.64	4.57 ns	4.55 ns	4.94	7.32 ^2
Relative liver weight (g/kg bw)	31.3	31.6 ns	32.4 ns	36.0 ^2	52.0 ^2
Liver histopathology	NSRE	NSRE	NSRE	NSRE	Hepatocyte hypertrophy, marked cytoplamic eosinophilia
Electron microscopy  of liver (number and morphology of peroxisomes an, and amount on smooth ER and in centrilobular and periportal hepatocytes )	NSRE	Not examined	Not examined	NSRE	Amount and size of peroxisomes marked increased peripotal and centrilobular, amount of smooth ER marked increase periportal and a week increase centrilobular
Total protein content in liver homogenat (mg/g liver)	121	217 ns	216 ns	214 ns	228 ^2
Microsomal protein content (mg/g liver)	33.7	33.4 ns	33.6 ns	32.8 ns	36.2 ns
Cyanide-insensitive palmitoyl-CoA oxidase activity in whole liver homogenate (nmol/min/mg protein)	5.63	6.16 ns	6.62 ^2	8.39 ^3	35.17 ^3
Lauric acid 11-hydroxylase activity in microsomal liver fraction (nmol/min/mg protein)	0.21	0.23 ns	0.27 ^1	0.29 ^1	0.64 ^1
Lauric acid 12-hydroxylase activity in microsomal liver fraction (nmol/min/mg protein)	0.36	0.40 ns	0.46 ^1	0.65 ^1	2.87 ^2

ns         non siginficant p >0.05

¹           significant p ≤ 0.05

²           significant p ≤ 0.01

³           significant p ≤ 0.001

NSRE   no substance related effects observed

Applicant's summary and conclusion

Executive summary:

This study was conducted according to OECD guideline 412, GLP standards were fulfilled. There are some minor deviations that are listed in the original final report. Overall the study is reliable without restriction.

The results of the present inhalation toxicity study with 2-ethylhexanal indicate that exposure of rats for four weeks affected growth, food conversion efficiency, differential white blood cells, clinical chemistry parameters, and the relative weight of all selected organs. The positive control di-(2-ethylhexyl)phtalate (DEHP) was included in this study as a positive control, because it was expected that 2-ethylhexanal is a peroxisome proliferator. DEHP resulted in increases in liver weight and peroxisome proliferation, hypolipidaemic action followed by decreased cholesterol and triglycerides levels in serum/plasma. DEHP was valid as a positive control for peroxisome proliferation in this study.

General toxic effects induced by 2-ethylhexanal consisted of reduced body weights and reduced food conversion efficiency in rats of both sexes. Rats at 250.7 ppm also showed effects of 2-ethylhexanal on the liver, increased relative liver weight, decreased plasma cholesterol, increased plasma triglycerides and decreased fasting glucose. In contrast, alkaline phosphatase (ALP) activity was increased at all concentration levels, although a clear dose-response relationship was not obtained. These changes were not accompanied by histopathological changes: The decrease in absolute and relative thymus weight as observed in rats exposed to 250.7 ppm 2-ethylhexanal (analytical concentration). However, there was no histopathological evidence of thymic atrophy.

A dose-related increase in both absolute and relative adrenal weight in exposed females was observed, together with a dose-related increase in relative kidney weight in these rats. Histopathological examination of these organs did not indicate treatment related effects of toxicity to 2-ethylhexanal. Decreased cholesterol levels were also seen in rats exposed to 250.7 ppm 2-ethylhexanal but their triglyceride level was increased. From the results of this study it was concluded that inhalation exposure of rats to 2-ethylhexanal for four weeks resulted in general toxicity (growth retardation and reduced food conversion efficiency), a decreased percentage of lymphocytes and increased percentage of neutrophils in blood, changes in weight of testes, adrenals, kidneys, thymus, heart and lungs and various changes indicative of hepatotoxicity at a level of 250.7 ppm.

Peroxisome proliferation: at the exposure levels examined, 2-EHN produced little effects on biochemical markers which could not be confirmed by qualitative structural analysis (histopathology) where no changes were observed at 250.7 ppm. Additionally, liver weight was not increased presenting a sensitive marker for peroxisome proliferation. Peroxisomal proliferators generally produce a more marked effect. 2-EHN is only a very weak peroxisome proliferator in this species. The relevance is questionable because such findings on peroxisome proliferation are usually restricted to rodents and are not important for human toxicity. Additionally, the observed effects can be explained by the metabolic product of 2 -ethylhexanal, 2 -ethylhexanoic acid (Deisinger et al., 1994), which is a known peroxisome proliferator. 2 -ethylhexanol is metabolized oxidative and quantitative to 2 -ethylhexanoic acid, 2 -ethylhexal is the aldehyde intermediate. There are no data available for 2 -ethylhxanal itself. Moreover, the positive control DEHP has been administered orally while 2EHN was administered via inhalation.

In summary, the exposure of rats to 2-ethylhexanal produced only small effects on relative liver weight at 25.5 ppm/0.14 mg/L and on the markers of hepatic peroxisome proliferation measured, this dose can be used as a NOEL for peroxisome proliferation. It may therefore be concluded that 2-ethylhexanal is only a very weak peroxisome proliferator in the rat with a NOAEL for overall effects/systemic toxicity of 102.2 ppm/0.54 mg/L (analytical concentration) can be derived.