3-methyl-1-isobutylbutyl 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:

25 September 2018 to 01 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1-isobutylbutyl acetate

EC Number:

233-588-4

EC Name:

3-methyl-1-isobutylbutyl acetate

Cas Number:

10250-45-0

Molecular formula:

C11H22O2

IUPAC Name:

3-methyl-1-isobutylbutyl acetate

Test material form:

liquid

Details on test material:

- Physical Description: Clear colourless liquid
- Storage Conditions: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™ (EPISKIN-SM™, 0.38 cm^2)
- Tissue batch number(s): 18 EKIN 039
- This model is a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.
- The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test material did not interfere with the MTT endpoint.

APPLICATION/ TREATMENT OF THE TEST MATERIAL
- Twenty-five μL of the undiluted test material was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5 % SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF REPLICATE TISSUES:
- The test was performed on a total of 3 tissues per test material together with negative and positive controls.

INTERPRETATION
- A test material is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50 % of the mean viability of the negative controls. The prediction to be considered is Category 2.
- A test material is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50 % of the mean viability of the negative controls. No category is required.

ANALYSIS
- Calculation of Cell Viability
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:

%Viability = (ODc/mean ODlt_u+MTT) * 100

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL
- Concentration: Undiluted

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration: 5 %:

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours followed by 3 hours with MTT

Number of replicates:

The test was performed on a total of 3 tissues per test material together with negative and positive controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20151202). Because no colour changes were observed it was concluded that the test material did not interact with the MTT endpoint.
- Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 76 %. Since the mean relative tissue viability for the test material was above 50 % it is considered to be non-irritant.
- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 32 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 14 %, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin irritation test

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570) ± SD
Negative control	0.963	0.925	1.034	0.974 ± 0.055
Test material	0.775	0.702	0.737	0.738 ± 0.037
Positive control	0.184	0.310	0.449	0.314 ± 0.132

OD = Optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability in the in vitro skin irritation test

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	5.7
Test material	76	3.8
Positive control	32	14

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

The objective of this study was to evaluate the test material for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM™)). The possible skin irritation potential of the test material was tested through topical application for 15 minutes.

The test material was applied undiluted (25 μL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test material.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 76 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.

The positive control had a mean cell viability of 32 % after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 14 %, indicating that the test system functioned properly.

Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.