Phenethyl isobutyrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-12 to 2015-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

2013

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:

Phenethyl isobutyrate

EC Number:

203-116-1

EC Name:

Phenethyl isobutyrate

Cas Number:

103-48-0

Molecular formula:

C12H16O2

IUPAC Name:

2-phenylethyl 2-methylpropanoate

In vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Justification for test system used:

The EpiDerm TM is an in vitro system recommended by the corresponding guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number: 21678
- Date of initiation of testing: 2015-06-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 35 minutes; room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing for 15 min, submerging in DPBS at least 3 times
- Observable damage in the tissue due to washing: none

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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.976 +/- 0.049 passed
- Barrier function: 6.44 hrs; passed
- Contamination: sterile; passed

NUMBER OF REPLICATE TISSUES: 3

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 viability after 60 minutes exposure and 42 h post exposure is less than or equal to 50% of the negative control
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure and 42 h post exposure is greater than 50% of the negative control

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 % solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h and 5 min

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

6.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Results

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. Absor-bance [%] Tissue 1, 2 + 3**	Relative standard deviation [%]	Rel. Absorbance [% of Negative Control]**
Negative control	60 min	1.417	1.382	1.413	1.404	100.9 98.4 100.7	1.4	100.0
Positive control	60 min	0.104	0.095	0.075	0.091	7.4 6.7 5.4	16.2	6.5
Test item	60 min	1.499	1.523	1.347	1.456	106.8 108.5 96	6.5	103.7

* Mean of three replicate wells after blank correction

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

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

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not considered to be irritant to skin.

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. 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 increased to 103.7% 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 substance is not irritant to skin.