Butyl ethyl 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:

2016-11-10 to 2016-12-22

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl ethyl ether

EC Number:

211-055-7

EC Name:

Butyl ethyl ether

Cas Number:

628-81-9

Molecular formula:

C6H14O

IUPAC Name:

1-ethoxybutane

Test material form:

liquid

Details on test material:

- Source and batch No.of test material: 60930023
- Expiration date of the batch: 09/2021
- Purity: 98.4%
- Physical State: liquid
- Density: 0.75 g/cm³
- Colour: colourless
- Storage condition of test material: Keep container tightly closed. Store in a dry and cool area

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS "Category 2". Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): 16-EKIN-046
- Expiration date: November 21, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable):
a) 37 ± 1 °C (5.0% CO2 for 42 ± 1 h) in 2 mL pre-warmed fresh maintenance medium
b) 37 ± 1 °C (5.0% CO2 for 3 h ± 5 min) in 2 mL pre-warmed MTT medium

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
* washed with DPBS; excess DPBS was removed by blotting bottom with blotting paper;
* after post-incubation: excess medium was removed by blotting bottom on absorbent paper

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2.
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered irritant to the skin if the viability after 15 minutes exposure and 42 h of post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure and 42 h is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm²) of the test item was dispensed directly atop the EPISKIN-SM tissue using a positive displacement pipette. The test item was spread to match size of the tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat. No. 14040-091, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277) in aqua dest.

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

73.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For detailed results see box "Any other information on results".

Any other information on results incl. tables

Results of the  Pre-Experiments

The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixtures of 10 µL of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC_living equalled 0%.

Table 1: Results of the test item butyl ethyl ether

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.760 0.824	0.715 0.735	0.741 0.795	0.235 0.182	0.176 0.180	0.185 0.153	0.573 0.573	0.532 0.523	0.607 0.618
OD570 (Blank corrected	0.717 0.781	0.672 0.693	0.698 0.752	0.192 0.139	0.133 0.137	0.142 0.110	0.531 0.530	0.489 0.480	0.565 0.575
Mean OD570 of the Duplicates (Blank corrected)	0.749	0.683	0.725	0.166	0.135	0.126	0.530	0.485	0.570
Total Mean OD570 of 3 Replicate Tissues (Blank corrected)	0.719*			0.142			0.528
SD OD570	0.034			0.021			0.043
Relative Tissue Viabilities [%]	104.2	94.9	100.9	23.0	18.8	17.6	73.7	67.4	79.3
Mean Relative Tissue Viabilities [%]	100.0			19.8**			73.5
SD Tissue Viabilities [%]***	4.7			2.9			5.9
CV [% Viability]	4.7			14.5			8.1

*               Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

**             Mean relative tissue viability of the three positive control tissues is ≤ 40%.

***           Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570Blank	0.043	< 0.1	pass
Mean Absolute OD570NK	0.762	0.6 ≤ NK ≤ 1.5	pass
Mean Relative Viability PC [%]	19.8	≤ 40%	pass
Max. SD of % Viability	5.9	≤ 18%	pass

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no irritant effects in an in vitro skin irritation study conducted according to OECD 439.

Executive summary:

In an in vitro skin irritation study according to OECD Guideline 439 (Reconstructed Human Epidermis Test), the potential of buthl ethyl ether (98.4% purity) to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM™ (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study the test item was applied topically to the EPISKIN-SM™ tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The test item showed no irritant effects and is therefore considered to be non-irritating to the skin.