1,3-dimethyl-3-phenylbutyl 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:

2017-04-28 to 2017-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

published 2003, 7h revision, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dimethyl-3-phenylbutyl acetate

EC Number:

268-407-8

EC Name:

1,3-dimethyl-3-phenylbutyl acetate

Cas Number:

68083-58-9

Molecular formula:

C14H20O2

IUPAC Name:

4-methyl-4-phenylpentan-2-yl acetate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes cultured from a multilayered, hughly differentiated model of the human epidermis

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: MatTek Corporation (Bratislava, Slovakia)
The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

- Procedure used: The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions and the gauze was removed and the inserts were taken out under an airflow using forceps. Any remaining agarose was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality.
- Quality control for skin discs: The tissues were checked for air bubbles between agarose and insert (should be less than 30% of the total surface), liquid on top of the insert was removed with sterile cotton tips, if again moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C,
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C,

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 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 and then 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: none reported
- Modifications to validated SOP: none reported

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Filter: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS AND DECISION CRITERIA
- The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability of three individual tissues after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test substance is considered to be non-irritant to skin 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

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL (47 μL/cm2)
- Concentration (if solution): test item was applied undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of DPBS was applied to triplicate tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL of SLS was applied to triplicate tissue.
- Concentration (if solution): 5% SLS solution in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

41 hours

Number of replicates:

Three replicates per test item, negative and positive controls were used.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

26.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

30.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

30.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

29.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

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 of water. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. Therefore, the test item passed the MTT- and the colour interference pre-tests.

The is considered valid as all the acceptability criteria were met:

In the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3% thus ensuring the validity of the test system.
The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 8% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 1. Results

Dose group	Tissue No.	Absorbance 570 nm			Mean absorbance of 3 wells	Mean absorbance of 3 wells blank corrected	Mean absorbance of 3 tissues	Rel. absorbance [%] tissue 1, 2+3	Rel. standard deviation	Mean Rel. absorbance [%]
		Well 1	Well 2	Well 3
Blank		0.038	0.038	0.037	0.037	0.000
Negative control	1	1.708	1.718	1.740	1.722	1.684	1.670	100.8	4.6	100.0
	2	1.804	1.757	1.767	1.776	1.738		104.1
	3	1.659	1.606	1.611	1.625	1.588		95.1
Positive control	1	0.090	0.095	0.094	0.093	0.055	0.055	3.3	0.6	3.3
	2	0.093	0.093	0.091	0.092	0.055		3.3
	3	0.092	0.093	0.093	0.093	0.055		3.3
Blank		0.037	0.037	0.036	0.037	0.000
Test item	1	0.489	0.473	0.477	0.480	0.443	0.485	26.5	7.5	29.1
	2	0.557	0.537	0.535	0.546	0.506		30.3
	3	0.555	0.540	0.536	0.544	0.507		30.4

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is considered as irritating to skin under test conditions as the mean relative absorbance value was reduced to 29.1% after exposure of the skin tissues compared to the relative absorbance value of the negative control.

Executive summary:

An in vitro study was performed to assess the irritation potential of test item by means of the Human Skin Model Test. The study was conducted according to OECD 439 and EU B.46 guidelines and is GLP-compliant.

Each three tissues of the human skin model EpiDerm were treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes.

After treatment with the negative control the absorbance values were well within 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. For the test item, the mean relative absorbance value decreased to 29.1% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, under the experimental conditions the test item is considered to be irritant to skin.

The study is reliable without restrictions (Klimisch 1) as it was conducted according to the standard methods and to GLP. It is therefore considered as relevant for key study for this endpoint.