p-methoxybenzyl acetate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-10-27 to 2015-11-23

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

p-methoxybenzyl acetate

EC Number:

203-185-8

EC Name:

p-methoxybenzyl acetate

Cas Number:

104-21-2

Molecular formula:

C10H12O3

IUPAC Name:

4-methoxybenzyl acetate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

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 onversion 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 Kit
- Tissue batch number: Lot No.: 23303
- Delivery date: 2015-11-17
- Date of initiation of testing: 2015-11-17 (pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times, volume not provided
- Observable damage in the tissue due to washing: no data provided
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive Contro: mean 4.77 %, range 4.00 % - 5.90 %

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
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 (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL (47 μL/cm2)
- Concentration: unchanged

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5 % SLS solution in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

post-treatment: 42 hours and 38 minutes
MTT: 3 h treatment and 69 h extraction

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

103.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.4 % tissue viability

Remarks on result:

no indication of irritation

In vivo

Other effects:

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

Any other information on results incl. tables

Dose Group	Exposure Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean rel. Absorbance [% of Negative Control]***
Negative control	1 h	1.402	1.549	1.502	1.484		5.1	100
Positive control	1 h	0.066	0.064	0.067	0.066		2.7	4.4
Test item	1 h	1.677	1.27	1.657	1.535		14.9	103.4

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100x(absorbance tissue)/ mean absorbance negative control

*** relative absorbance [rounded values]: (100x(mean absorbance test item/positive control))/ mean absorbance negative control

 

The mean relative absorbance value of the test item, corresponding to the cell viability, was 103.4 % (threshold for irritancy:≤50 %), consequently the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be non-irritant in the in vitro human skin model test with EpiDerm.

Executive summary:

The in vitro Human Skin Model Test with EpiDerm study according to OECD 439 was performed to assess the irritation potential of the test substance. Independent triplicate tissues of EpiDerm were exposed to either the test item, the negative control (DPBS) or the positive control (5 % SDS in water) for 1 hour. 30 μL of the test item were dispensed directly onto triplicate EpiDerm tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test substance the mean tissue viability was 103.4 % after 1 h exposure. This value is above the threshold for irritation, which is defined to be < 50 %. Therefore, the test substance was considered to be not skin irritating. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test was not irritating to skin according to EU CLP and UN GHS.