4-tert-Butoxystyrene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

13 April 2004

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(tert-butoxy)-4-ethenylbenzene

EC Number:

619-127-3

Cas Number:

95418-58-9

Molecular formula:

C12H16O

IUPAC Name:

1-(tert-butoxy)-4-ethenylbenzene

Test material form:

liquid

Details on test material:

Clear and colourless

In vitro test system

Test system:

human skin model

Cell source:

other: MatTek Corporation, Ashland MA, USA

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): 9640 kit T
- Storage and preparation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. The next day, at least one hour before the assay is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The medium was replaced with fresh DMEM medium just before butoxystyrene was applied.

ENVIRONMENTAL CONDITIONS
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C
- Humidity: 80-100%.
Temporary deviations from the humidity (with a maximum of 8%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. It is only a minor deviation in the humidity and the OD540 of the negative and positive controls were all within the historical data range. Therefore this deviation has no effect on the results of the study.
- CO2 percentage in the air: 5%
- Photoperiod: dark

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 h
- Spectrophotometer: Multiskan Spectrum (Thermo Labsystems)
- Wavelength: 540 nm

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µl of undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied: 50 µl of Milli-Q water

POSITIVE CONTROL
- Amount(s) applied: 50 µl
- Concentration: 8N potassium hydroxide

Duration of treatment / exposure:

Two time periods were used for exposure to the test substance: 3 minutes and one hour.

Duration of post-treatment incubation (if applicable):

For cell viability measurement, tissues were incubated for 3 h at 37 °C in air containing 5 ± 0.5% carbon dioxide.

Number of replicates:

The test was performed on a total of 4 tissues per test substance (two for 3 min exposure and two for 1 h exposure) together with a negative control and positive control for each exposure period.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

The relative mean tissue viability obtained after 1 hour treatment with butoxystyrene compared to negative control tissues.

Value:

121

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

The relative mean tissue viability obtained after 3 minutes treatment with butoxystyrene compared to negative control tissues.

Value:

112

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The absolute mean optical density at 540 nm of the two tissues of the negative control was within the laboratory historical control data range.
- Acceptance criteria met for positive control:
The mean relative tissue viability of the 3 minutes exposure of the positive control was 4% (should be < or = 30%).
- Acceptance criteria met for variability between replicate measurements:
a) The maximum inter tissue variability (in viability) was less than 3% between two tissues treated identically (should be max. 30%).
b) The maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2% (should be max. 15%).

It was therefore concluded that the test system functioned properly.

Any other information on results incl. tables

The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with butoxystyrene compared to the negative control tissues was 112% and 121% respectively.

It was noted that butoxystyrene had affected the cell culture insert of the skin tissue after exposure.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Butoxystyrene is not corrosive in the in vitro skin corrosion test under the described experimental conditions.

Executive summary:

The corrosive properties of butoxystyrene on a human three dimensional epidermal model (EpiDerm (EPI-200)) were studied. The possible corrosive potential of butoxystyrene was tested through topical application for 3 minutes and 1 hour. 

Butoxystyrene was applied undiluted (50 µl) directly on top of the skin tissue. The positive control had a mean relative tissue viability after 3 minutes exposure of 4%. The absolute mean OD₅₄₀ (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter tissue variability in viability between two tissues treated identically was less than 3% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with butoxystyrene compared to the negative control tissues was 112% and 121% respectively. Since the mean relative tissue viability for butoxystyrene was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment butoxystyrene is considered to be not corrosive.