Isobutyl 2-naphthyl ether

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May, 2016 - 17 May, 2016

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)

Version / remarks:

28 July 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:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Isobutyl 2-naphthyl ether

EC Number:

218-529-2

EC Name:

Isobutyl 2-naphthyl ether

Cas Number:

2173-57-1

Molecular formula:

C14H16O

IUPAC Name:

isobutyl 2-naphthyl ether

Test material form:

other: solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-019
- The solid test item was melted before use and twenty five μL of the undiluted test substance (liquid) was added into 12-well plates on top of the skin tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 34.9 - 37.0°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test item by assessment of its effect on a three dimensional human epidermis
model. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
A test item 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 item is present on the tissues when the MTT viability test is performed.

Test for Direct MTT Reduction + colour interference:
Naph Iso Butyl Ether Beta was checked for possible direct MTT reduction and colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, 10 μL of Naph Iso Butyl Ether Beta was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 24 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
The test was performed on a total of 3 tissues per test item together with negative and positive controls.
Twenty five μL of the undiluted test item 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. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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.

CELL VIABILITY MEASUREMENT:
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 h 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 prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 71 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

DECISION CRITERIA
- A test substance 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 substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance 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 substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material
- Applied volume: 25 μL

Duration of treatment / exposure:

15-Minute exposure period and 42 hours post-exposure incubation period.

Number of replicates:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: relative mean viability

Remarks:

(%)

Value:

89

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability compared to the negative control tissues (100%).

Other effects / acceptance of results:

Direct MTT Reduction:
Naph Iso Butyl Ether Beta was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that the test item did not interact with the MTT endpoint.
Test Item, Positive Control Item and Negative Control Item
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Naph Iso Butyl Ether Beta compared to the negative control tissues was 89%. Since the mean relative tissue viability for Naph Iso Butyl Ether Beta was above 50% Naph Iso Butyl Ether Beta is considered to be non-irritant.
Quality Criteria
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 12% relative to the negative control. 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 less than 13% for the negative control, positive control and the test item, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD562 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	0.755	0.882	0.112		100
	0.924
	0.968
Positive Control Item	0.115	0.107	0.035	15	12
	0.136			15
	0.069			7
Test Item	0.673	0.784	0.096	89	89
	0.844			91
	0.836			86

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Applicant's summary and conclusion

Interpretation of results:

other: Not skin irritating.

Remarks:

According to Regulation (EC) No. 1272/2008 and its updates.

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 89%. Since the mean relative tissue viability for the substance was above 50%, the substance is considered to be non-irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. The substance was melted before application (melting point is 32oC). After 42 hours post-exposure 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 12% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 13% for the negative control, positive control and the test item, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 89%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be non-irritant.