2-ethylhexyl acrylate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Apr 1985 - 19 July 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

yes

Remarks:

food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl acrylate

EC Number:

203-080-7

EC Name:

2-ethylhexyl acrylate

Cas Number:

103-11-7

Molecular formula:

C11H20O2

IUPAC Name:

2-ethylhexyl acrylate

Details on test material:

- Name of test material (as cited in study report): 2-ethylhexyl acrylate
- Physical state: liquid
- Analytical purity: 99.7 %
- Substance No.: 85/81
- Stability under test conditions: The stability of the test substance during the study period was assessed by reanalysis (BASF AG, ZHU Report from 02 Sep 1985).
- Storage condition of test material: refrigerator

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach, Germany
- Age at study initiation: approximately 8-week old
- Weight at study initiation (mean): male rats: 223 g (213 - 234 g); female rats: 158 g (150 - 164 g)
- Housing: single
- Diet (ad libitum): Kliba Labordiaet Ratte/Maus A 343 10 mm Pellet, Klingentalmuehle AG, CH-4303 Kaiseraugst
- Water (ad libitum): tap water
- Acclimation period: 5 days


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

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chambers (glass/steel inhalation chambers) with a capacity of approx. 1.3 m3
- Method of holding animals in test chamber: animals in wire cages
- Temperature, humidity in air chamber: 22.8-23.6 °C, 36-41 %


TEST ATMOSPHERE
- Brief description of analytical method used: The test substance concentrations were checked intermittently (10 and 30 ppm) or continuously (100 ppm) by total hydrocarbons analyzers (THCA)/GC/FID.
- Samples taken from breathing zone: yes

EXPOSURE SYSTEM
The test and control animals were exposed to the test substance vapors and control air in a whole-body exposure system, respectively. All air streams, supply air and exhaust air of all test groups were adjusted by air flow meters (rotameters), measured continuously and recorded approximately every 2 hours. The air temperatures in the exposure systems were measured continuously with multipurpose thermometers (Diehl GmbH & Co) and recorded approximately every 2 hours. The pressures in the exposure systems were measured continuously (slanting tube pressure gage) and recorded once a day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Measured test substance concentrations were within ± 5% of the nominal concentrations (see table below).

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 h/day; 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

0.075 mg/L

Doses / concentrations 2

Dose / conc.:

30 ppm

Remarks:

0.225 mg/L

Doses / concentrations 3

Dose / conc.:

100 ppm

Remarks:

0.753 mg/L

No. of animals per sex per dose:

Groups of 10 male and 10 female rats per test concentration were exposed to test substance vapors in a whole-body exposure system on 6 hours per day, 5 days per week at 10, 30, and 100 ppm (corresponding to approximately 0.075 mg/L, 0.226 mg/L or 0.753 mg/L).

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In order to obtain preliminary information about the profile of action of 2-EHA vapor, the maximum concentration during the 90-day study was to be within the saturation range of the test substance (estimated by extrapolation of the vapor-pressure curve: T = 20°C; p= 0.13 mbar = 130 ppm). For technical reasons (e.g. condensation phenomena and hence poor reproducibility with slight temperature fluctuations), the maximum concentration which could be reliably maintained under the study conditions was chosen to be 100 ppm. The minimum concentration of 10 ppm was based on the applicable MAC value for methyl acrylate (1984). The intermediate concentration was established between these two concentrations as 30 ppm in order to obtain any concentration-response relationships.

- Post-exposure period: none

- Control: An air control with 10 male and 10 female rats was run in parallel.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Mortality and clinical signs were checked each day.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weight determinations were conducted once a week.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals in the control group and the high dose group were checked with a focusable hand-held slit lamp for changes in the refracting media at the beginning of the pre-flow period (day -8) and at the end of the study (day 83).
- Dose groups that were examined: high-dose, control


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- Anaesthetic used for blood collection: No
- How many animals: 10
- Parameters were examined:
The following hematological examinations were carried out on 10 animals per test group and sex at the end of the exposure period: Hemoglobin, erythrocytes, hematocrit, mean hemoglobin content per erythrocyte, mean cell volume, mean corpuscular hemoglobin concentration, platelets, leukocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the exposure period
- How many animals:
- Parameters were examined:
The following clinicochemical examinations were carried out on 10 animals per test group and sex at the end of the exposure period: Total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, inorganic phosphate, calcium, chloride, triglycerides, cholesterol, albumine, globulins.
The following enzyme activities were determined: - Glutamic-pyruvic transaminase - Alkaline phosphatase - Glutamic-oxalacetic transaminase. Clotting analyses were performed: Prothrombin timne (Hepato Quick's test).


URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The animals were sacrificed at the end of the 3-month inhalation period and each animal was completely necropsied. Histopathologic examination was carried out on 31 organs/tissues of high dose and control animals. The lungs, nasal cavity, thyroid and parathyroid glands, trachea and liver from all animals/all test groups were subjected to histopathology examination.

Statistics:

T-test according to Williams.

WILLIAMS DA (1971 ). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103 - 117
WILLIAMS DA (1972 ). The comparisdn of several dose levels with a zero dose control. Biometrics 28: 519 - 531

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Mild lethargy, eyelid closure during exposure.
- 30 ppm (0.225 mg/L): Mild lethargy and eyelid closure during exposure.
- 10 ppm (0.075 mg/L): As compared to control animals, no toxic effects which could be interpreted as being related to the test substance.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Retarded body weight gain in both sexes on determination of the body weight change; retarded body weight gain in male animals only on determination of body weight.
- 30 ppm (0.225 mg/L): Slight retardation in body weight gain of the female animals.
- 10 ppm (0.075 mg/L): As compared to control animals, no toxic effects which could be interpreted as being related to the test substance.

Food consumption and compound intake (if feeding study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Increase in glutamic-pyruvic transaminase activity and alkaline phosphatase activity of female rats; reduction of total proteins in female animals and very slightly in male rats; slightly reduced albumin concentrations in both sexes (not statistically significant); reduction of glucose level in female animals.
- 30 ppm (0.226 mg/L): Reduced total protein and albumin levels in both sexes; only a tendency to a reduction in albumin levels in female animals.
- 10 ppm (0.075 mg/L): As compared to control animals, no toxic effects which could be interpreted as being related to the test substance.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 100 ppm (0.753 mg/L): Reduction in body weight resulted in darker coloration of liver parenchyma with indistinct lobular marking (decrease in liver lipids).
- 30 ppm (0.226 mg/L): No findings.
- 10 ppm (0.075 mg/L): No findings.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nasal cavity:
Degeneration of olfactory mucosa was diagnosed. This degeneration was characterized by a reduction of cellular layers, reduction or loss of apical cytoplasmic structures such as olfactory knobs and microvilli, and by necrosis. Identification of the remaining olfactory mucosa cells was not possible. Occasional mitoses were present.
Level I: Degeneration of olfactory mucosa was diagnosed in all rats of 100 ppm group and in 4 males and 4 females each of 30 ppm group. In 100 ppm group, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. In 30 ppm group, the degeneration affected only small areas of the dorsolateral olfactory mucosa and was minimal in severity.
LeveL II: Degeneration of olfactory mucosa was diagnosed in all rats of 100 ppm group, 1 male of group 2, 2 males and 1 femaLe of 10 ppm group 1, and 1 male of control group. In 100 ppm group, the degeneration was diffuse (dorsal, dorsolateral), whereas in the other groups, the degeneration was focal. The severity was minimal to marked in 100 ppm group, and mainly minimal in the other groups.
LeveL III: Degeneration of oLfactory mucosa was only diagnosed in one male and one female each of the 100 ppmgroup. The severity was slight.
Level IV: No degeneration of the olfactory mucosa was noted.

Liver
Fatty change, characterized by micro- to macrovesicular hepatocellular fat deposits was diagnosed in rats of all groups including controls.
The fatty change was mainly localized in zones 1 (periportal zone) and 2 (intermediate zone). In 5 males of 30 ppm group and in one male of 100 ppm group, fatty change was noted also in zone 3 (zone surrounding terminal hepatic venule). Generally, the severity of fatty change was higher in males than in females. In females, the severity of fatty change was similar in all groups. In males, the severity of fatty change was less in 100 ppmgroup when compared with the rats of the other groups.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

nasal irritation

Effect level:

0.075 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

0.226 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

0.225 mg/L air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Concentrations in the inhalation chambers:

A study mean with standard deviation was calculated from the daily means of the individual chamber concentrations of the test groups:
		Concentration
     Nominal		       Measured
 [ppm]	[mg/L]		[ppm]    	[mg/L]
----------------------------------------------
  10	0.075		9.9 ± 0.47	0.0745
  30    0.226		30 ± 1.7	0.226
 100    0.753		100 ± 4.6	0.753
----------------------------------------------

Applicant's summary and conclusion

Executive summary:

In a valid 90-day inhalation study (BASF, 1989) Wistar rats were administered in a whole-body exposition on 6 hours per day, 5 days per week, to 2-EHA vapour at concentrations of 0 ppm, 10 ppm, 30 ppm or 100 ppm (approximately 0.075 mg/l, 0.226 mg/l or 0.753 mg/l for the treatment groups) (2-EHA purity 99.7%). The study design was conducted according to OECD 413 (1981). Compared to the actual version of the test guideline, validity is restricted in that food consumption was not recorded, lung tissues were not perfused, and laryngopharynx was not examined. Histopathologic examination was carried out on 31 organs/tissues of high dose and control animals. The lungs, nasal cavity, thyroid and parathyroid glands, trachea and liver from all animals/all test groups were subjected to histopathology examination.

There were no treatment-related premature deaths. During exposure period animals of the high and mid dose groups exhibited lethargy and ptosis. Body weight gain was lower in both sexes of the high dose during and at the end of the study. A transiently reduced body weight gain was observed in mid dose females. From day 21 onwards mean body weight (absolute) was lower in high dose males compared to the control group. This parameter was not significantly altered in any other group at any time point during the study. Activities of ALAT and alkaline phosphatase were elevated in high dose females. In high dose males and females lower levels of total protein, albumin and glucose were demonstrated. Reduced protein and albumin values were also seen in each sex of the mid dose groups.

Absolute liver weight was reduced in high dose males and relative adrenal weights were lower in high dose males and females compared to the control groups. The microscopic examination revealed no lesion other than a focal or diffuse degeneration of the olfactory epithelium of the cranial nasal cavity in animals of both sexes of the high and mid dose groups. All rats of the 100 ppm group showed degeneration of the olfactory mucosa in the anterior part of the nasal cavity. The incidence of degeneration of the olfactory mucosa but not the severity was increased in mid dose rats. No treatment-related lesion of the nasal cavity was diagnosed at the low dose level.

Degeneration of the olfactory epithelium was characterised by a reduction of cell layers, reduction or loss of apical cytoplasmic structures such as olfactory knobs and microvilli, and by necrosis. Identification of the remaining olfactory mucosa cells was not possible. Occasional mitosis were present.

In detail, degeneration of the olfactory mucosa was diagnosed in the anterior part of the nasal turbinates (level 1) in all high dose rats and in four males and four females of the mid dose group. At the high dose level, the degeneration affected the olfactory mucosa diffusely in the dorsal and dorsolateral area, and the severity was mainly moderate. Mid dose animals showed small areas of degeneration of the dorsolateral olfactory mucosa of minimal severity. At level 2 of the turbinates, degeneration was diagnosed in all high dose rats, one mid dose male, two female and one female of the low dose group, and in one control group male. In the high dose group, the degeneration was diffuse in the dorsal and dorsolateral region, whereas in the other groups the degeneration was focal. The severity was minimal to marked in the high dose group, and mainly minimal in the other groups. Slight degeneration of the olfactory mucosa of the level 3 was only diagnosed in one male and one female each of the high dose group. 2-EHA induced no lesions of the trachea and the lungs, data of the pharynx/larynx were not available.

Table   2-EHA induced olfactory degeneration in rats from a 90-day inhalation study (BASF, 1989)

 

	Dose group	Control		10 ppm		30 ppm		100 ppm
	Sex	M	F	M	F	M	F	M	F
	No. of animals	10	10	10	10	10	10	10	10
Nasal cavity	Level 1 (anterior)					4	4	10	10
	Mean severity grade					1	1	2.9	3.1
	Level 2	1		2	1	1		10	10
	Mean severity grade	1		1	1	2		2.9	2.5
	Level 3							1	1
	Mean severity grade							2	2

Grading used  1 = minimal, 2 = slight, 3 = moderate, 4 = marked; M = male, F = female

Treatment-related microscopic lesions outside the respiratory tract were seen in the liver. Fatty change (at low and medium severity grades a common finding in well fed rats) occurred in rats of all dose groups and control groups. In high dose males, the severity of fatty change was less compared to other groups and the control. The mean severity grades of lipid accumulation in the periportal zone of the liver lobus decreased from 2.6 in control males to 1.0 in the high concentration males, a minimal change was also seen in high concentration females (1.0 versus 1.6 in controls). No indication of a peroxisomal proliferation was evident in electron microscopy. No treatment-related effects were evident at the low dose group.

Reduced body weight gain, lower levels of parameters of the protein metabolism, the reduced serum glucose concentration and the reduced lipid accumulation in liver cells were assumed to be induced by a lower food consumption possibly resulting from the irritation effect on the respiratory tract of exposed animals.

The No-observed-adverse-effect-level (NOAEL) for local effects on the respiratory tract was 10 ppm (and 0.075 mg/l), and the NOAEC for systemic toxic effects was 30 ppm (and 0.226 mg/l).