3-methylbutyl butyrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-22 to 2015-02-16

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:

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

Deviations:

yes

Remarks:

without any impact on the outcome of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

without any impact on the outcome of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbutyl butyrate

EC Number:

203-380-8

EC Name:

3-methylbutyl butyrate

Cas Number:

106-27-4

Molecular formula:

C9H18O2

IUPAC Name:

3-methylbutyl butanoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm TM tissues (Epi-200-SIT Kit)

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: The test substance was added into the insert atop the corresponding EpiDerm™ triplicate tissues
- Amount/test concentration: 30 µL
- Duration of treatment: 60 minutes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1 °C in the remaining period at room temperature
- Temperature of post-treatment incubation: 37 ± 1 °C

CONTROL
- Negative Control: 30 µL DPBS (Gibco) was used as negative control per tissue;
- Positive Control: 30 µL of a 5 % SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: Tissues washed 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

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

- Viability:
Positive control: mean viability: 5.6 %; relative standard deviation: 2.4 %; range of viabilities: 2.9 - 11.3 %.
-Absorption:
Positive control: mean absorption: 0.098; relative standard deviation: 0.038 %; range of absorbance: 0.030 - 0.194
Negative control: mean absorption: 1.796; relative standard deviation: 0.281%, range of absorbance: 1.397 - 2.651

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Yes. Evaluation of colour interference: 30 µL of the test item mixed with 300 µL deionised water was incubated for 60 minutes at 37 ± 1.5 °C. Since the colour of the test item mixture did not change during the incubation period, additional testing with viable tissues was not required.
Evaluation of direct reduction of MTT: 30 µL of the test item added to MTT (1 mg/mL). Incubated in the dark at room temperature for 60 minutes. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50 %; irritant (I), H315 (category 2)
mean tissue viability > 50 %; non-irritant (NI)
Amount/concentration applied :undiluted
Duration of treatment / exposure: 60 minutes

Acceptability of the Assay
Criterion 1 (negative control): The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is > 0.8 and = 2.8.
Criterion 2 (positive control): An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is at or below 20 %.
Criterion 3 (standard deviation): The SD of 3 identical replicates should be < 18 %.
OD values should not be below historically established boundaries.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

115.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean relative absorbance

Other effects / acceptance of results:

- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT: test item did not change colour in the colour interference test
- 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; the mean relative absorbance value of the positive control was 3.8 % compared to the negative control.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions reported, the test substance is not irritating to skin.

Executive summary:

The skin irritant potential of the test item was determined by means of the Human Skin Model Test (OECD 439). The relative absorbance value of the test item was 115.2% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of <= 50%. There, the test item is not considered to possess an irritant potential.