2-ethylhexyl acrylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June 2002 - 29 Nov 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study conducted in compliance with GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

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
- Chemical name: 2-propenoic acid, 2-ethylhexyl ester
- Test substance No.: 00/0903-2
- Batch/Lot No.: Aus Behaelter B 1035 (Reinfallgefaess 1)
- Analytical Purity: 99.9 %
- Purity test date: 04 June 2002 (BASF AG, Analytical report)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar (CrlGlxBrlHan:Wl)
- Source: Charles River Deutschland GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: about 229 g (mean)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: single
- Diet (ad libitum): Standardized pelleted feed (Ratte - Maus - Hamster Diaet, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (ad libitum): drinking water


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, corn oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo UDS assay and for which historical data are available.
- Concentration of test material in vehicle: 10 and 20 g/100 mL, respectively
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Dose volume was 10 mL/kg bw of a solution with a concentration of 10 g/100 mL and 20 g/100 mL, respectively.

Duration of treatment / exposure:

single

Frequency of treatment:

Male animals per sacrifice interval were treated once per gavage and livers were perfused 3 hours and 14 hours after treatment, respectively. No. of animals per dose: 3 animals per group; two groups per dose level and control. In one group perfusion of livers was performed 3 hours and in the other group 14 hours after treatment, respectively.

Post exposure period:

no

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene (2-AAF)
- Route of administration: oral: gavage
- Doses / concentrations: 50 mg/kg bw suspended in corn oil in a volume of 10 mL/kg bw

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline were survived by all animals (male and female) without any clinical signs. Thus, only male animals were used for the main experiment and a doseof 2000 mg/kg body weight was selected as the highest dose; 1,000 mg/kg body weight were administered as further dose.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Liver perfusion and preparation of the primary rat hepatocytes: The livers were perfused with ethylene glycol-bis(beta-amino ethyl ether) N,N,N',N',-tetraacetic acid (EGTA) solution followed by collagenase solution. After removal of the livers and isolation of the hepatocytes, the cells were washed with about 20 mL Williams medium E incomplete (WMEI) (about 20 °C) and mixed thoroughly with a pipette. The cell suspension was then filtered using sterile gauze. After centrifugation (500 rpm) for about 5 minutes, the supernatant was discarded and the pellet resuspended in about 20 ml WMEI to yield single cell suspensions.


DETERMINATION OF CELL VIABILITY
The viability of the hepatocytes was determined by the trypan blue exclusion method. In addition, the number of the isolated cells was determined. It was also examined whether significant morphological changes of the cells or a reduction of the cell material occurred after test substance treatment.


SEEDING AND ATTACHMENT
The isolated hepatocytes were seeded on coverslips on 1.9 cm2 wells containing 2 mL of attachment medium. About 400000 viable cells were seeded per well. 6 wells per animal were used for the UDS assay. After an attachment period of about 2 hours with 5 % CO2 at 37 °C and >= 90 % humidity, the medium was replaced by fresh medium to remove non-adherent cells.


LABELING
The medium was replaced by 2 mL labeling medium, and the cells were incubated with 5 % C02 at 37 °C and >= 90 % humidity for 4 hours. After the labeling period, cells were washed; then fresh medium containing 0.25 mM unlabeled thymidine was added and the cells were incubated for another 14 hours. The cells on the coverslips were then fixed with ethanol/acetic acid (3 : 1, v/v) for at least 30 minutes, rinsed 2-4 times with aqua dest. and air-dried. The dried coverslips were mounted cell side up on glass slides using Corbit-Balsam and dried overnight.


AUTORADIOGRAPHY
The slides were coated with KODAK NTB-2 photographic emulsion (at about 37 °C) for about 5-10 seconds. After drying at room temperature in the dark (lightproof boxes), the coated slides were stored in the dark with a desiccant at -20 °C for 3-12 days. Thereafter, the slide boxes were left at room temperature for at least 3 hours. The photographic emulsion was then developed with KODAK D-19 (about 15 °C), fixed in KODAK Acidofix for about 5 minutes, washed in water for about 5-10 minutes and stained with methyl green-pyronine Y. After rinsing with water and ethanol and air-drying, the slides were covered with a 2nd coverslip using Corbit-Balsam.


QUANTIFICATION OF UDS/MICROSCOPIC EVALUATION
The quantification of UDS was performed microscopically using 2 or 3 slides per test group. 25-50 cells in good morphological conditions were randomly selected per slide and examined to achieve a total number of 100 cells/animal.
For each cell, the following counts were performed with an automatic image analyzer:
- the nuclear grain (NG) count (= number of silver grains overlying the nucleus)
- the cytoplasmic grain (CG) count (= number of grains in two or three nucleusequivalent areas adjacent to the nucleus).

Evaluation criteria:

The following parameters were calculated:
-the net nuclear grain (NNG) count of each cell (= nuclear grain count minus cytoplasmic grain count; NG - CG)
- the mean nuclear grain (NG) count
- the mean cytoplasmic grain (CG) count - the mean net nuclear grain (NNG) count
- the percentage of cells in repair (cells showing net nuclear grain counts of >= 0) - the percentage of cells in repair (cells showing net nuclear grain counts of >= 5)

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The substance 2-Ethylhexylacrylate was tested for its ability to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in vivo in rat hepatocytes. For this purpose, the test substance, dissolved in corn oil, was administered once orally to male Wistar rats at dose levels of 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. Hepatocytes were harvested 3 and 14 hours after administration of the test substance.

Clinical examinations:
The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. The administration of the test substance did not lead to signs of toxicity. The single administration of the positive control substance 2-AAF in a dose of 50 mg/kg body weight did not cause any evident signs of toxicity.

Cell viability:
Cell viability was not influenced by test substance treatment. Morphological changes of the cells were not obsereved, cell material was not reduced.

DNA repair activity:
As a negative control, male rats were administered merely the vehicle, corn oil, by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range. The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis. On the basis of the results from the present study, the single oral treatment with the test substance did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes.

Any other information on results incl. tables

Perfusion 3 h after treatment:

						Test groups

Mean per group		vehicle		1000 mg/kg	2000 mg/kg	positive
 +/- SD			control						control
-------------------------------------------------------------------------------------
NG counts		6.65+/-0.86	6.14+/-0.52	5.93+/-0.71	22.89+/-5.15

CG counts		12.29+/-0.60	10.59+/-0.42	10.37+/-1.08	12.45+/-1.14

NNG counts		-5.64+/-0.27	-4.45+/-0.26	-4.44+/-0.43	10.44+/-4.43

% cells in repair	8.67+/-4.93	10.00+/-3.61	8.00+/-2.00	89.33+/-8.08
(NNG >= 0)

% cells in repair	0.33+/-0.58	0.00+/-0.00	0.00+/-0.00	72.67+/-17.21
(NNG >= 5)
-------------------------------------------------------------------------------------


Perfusion 14 h after treatment:

						Test groups

Mean per group		vehicle		1000 mg/kg	2000 mg/kg	positive
 +/- SD			control						control
-------------------------------------------------------------------------------------
NG counts		6.64+/-0.33	7.73+/-0.64	7.61+/-0.63	23.50+/-3.40

CG counts		11.45+/-1.41	11.14+/-1.25	12.60+/-0.35	11.76+/-1.14

NNG counts		-4.99+/-1.10	-3.41+/-1.30	-4.98+/-0.36	11.75+/-2.34

% cells in repair	6.33+/-3.21	18.00+/-10.39	9.33+/-0.58	94.00+/-2.00
(NNG >= 0)

% cells in repair	0.00+/-0.00	1.00+/-1.00	0.00+/-0.00	77.33+/-10.26
(NNG >= 5)
-------------------------------------------------------------------------------------
NG = nuclear grains
CG = cytoplasmic grains
NNG = net nuclear grains


Thus, under the experimental conditions of this assay, the test article 2-Ethylhexyl acrylate is considered to be negative in the in vivo UDS assay using rat hepatocytes.

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

2-Ethylhexylacrylate was tested for its ability to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in vivo in rat hepatocytes. For this purpose, the test substance, dissolved in corn oil, was administered once orally to male Wistar rats at dose levels of 1,000 mg/kg and 2,000 mg/kg body weight in a volume of 10 ml/kg body weight in each case. Hepatocytes were harvested 3 and 14 hours after administration of the test substance. As a negative control, male rats were administered merely the vehicle, corn oil, by the same route, which gave frequencies of mean nuclear net grain counts within the historical control range. The positive control chemical 2-acetylaminofluorene (2-AAF) administered once orally in

a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis.

On the basis of the results from the present study, the single oral treatment with the test substance did not lead to an increase in the mean number of net nuclear grain counts at any dose level or exposure time in rat hepatocytes.

2-Ethylhexylacrylat is considered to be negative in the in vivo UDS assay using rat hepatocytes.