Methyl phenylacetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-11-09 to 2016-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl phenylacetate

EC Number:

202-940-9

EC Name:

Methyl phenylacetate

Cas Number:

101-41-7

Molecular formula:

C9H10O2

IUPAC Name:

methyl phenylacetate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT kits and MTT-100 assays
- Tissue batch number(s): Lot No.: 23305
- Shipping date: 01 December 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). A volume of 300 μL of the MTT solution was added to each well and the plates were kept in an incubator.
- Incubation time: 3 h (37 ± 1 °C, 5 ± 0.5 % CO2)
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 ± 1 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose 30 μL of the test item were mixed with 300 μL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C (5 ± 0.5% CO2). The colour of test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed. For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. For this purpose, approx. 30 μL of the test item were added to 1 mL of MTT solution (MTT (Sigma, Germany) concentrate diluted with MTT diluent (DMEM, Gibco, Germany) (resulting: 1 mg/mL)). This mixture was incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 60 minutes. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item according to EU classification H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test item: 30 µL (undiluted)
Negative control: 30 µL DPBS)
Positive control: 30 µL of a 5 % Sodium Lauryl Sulphate solution in deionised water

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours and 18 minutes

Number of replicates:

3

Test animals

Details on test animals or test system and environmental conditions:

not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

78.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The experiment was performed twice. Because the validity criteria of the test system were not met in the first experiment, the results of the first experiment are not used for evaluation and are not reported. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. 30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value was reduced irrelevantly to 78.2% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.