Ethyl hexanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-12 to 2015-07-17

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.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

yes

Remarks:

without any impact on the outcome of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) 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:

Ethyl hexanoate

EC Number:

204-640-3

EC Name:

Ethyl hexanoate

Cas Number:

123-66-0

Molecular formula:

C8H16O2

IUPAC Name:

ethyl hexanoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm Ø).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: EpiDerm tissues were treated with the test substance
- Amount/test concentration: 50 µL
- Duration of treatment: 3 minutes, 60 minutes
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure : room tempeature
- Temperature of post-treatment incubation (if applicable): 37+-1.5°C
CONTROL
- Negative Control: 50 µL deionised water was used as negative control per tissue;
- Positive Control: 50 µL 8.0 N potassium hydoxide (Sigma) was used a positive control per tissue
REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing (if done): Tissues washed with DPBS at least 20 times in order to remove any residual test material. Exess DPBS was removed by gently shaking the tissue inserts and blotting the lower surface with blotting paper.
SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50% after 3 minutes exposure; corrosive: optional sub-category 1A
mean tissue viability = 50% after 3 minutes exposure AND < 15% after 60 minutes exposure; Corrosive: Optional Sub-category 1B and 1C
mean tissue viability = 50% after 3 minutes exposure AND = 15% after 60 minutes exposuref; Non-corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

undiluted

Duration of treatment / exposure:

3 minutes; 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

109

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

108.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT:it did not change colour

Any other information on results incl. tables

Dose Group	Exposure Interval	Absorbance Well 1 (Tissue ½)	Absorbance Well 2 (Tissue ½)	Absorbance Well 3 (Tissue ½)	Mean Absorbance (Tissue ½)	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Mean Absorbance of 2 Tissues	CV [%]	Rel Absorbance [% of Negative Control]**
Negative control	3 min	1.642 1.694	1.670 1.529	1.339 1.637	1.550 1.620	1.511	1.580	1.546	3.2	100.0
Positive control		0.442 0.381	0.422 0.421	0.395 0.352	0.420 0.384	0.380	0.345	0.363	6.9	23.5
Test item		1.772 1.651	1.744 1.659	1.875 1.644	1.797 1.651	1.757	1.612	1.685	6.1	109.0
Negative control	60 min	1.488 1.603	1.654 1.612	1.649 1.598	1.597 1.604	1.560	1.568	1.564	0.3	100.0
Positive control		0.203 0.184	0.207 0.181	0.185 0.201	0.198 0.189	0.162	0.152	0.157	4.3	10.0
Test item		1.800 1.631	1.775 1.692	1.799 1.714	1.791 1.679	1.754	1.642	1.698	4.7	108.6

* Mean of three replicates wells after blank correction

** relative absorbance [rounded values]: 100 * (absorbance test item / positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the test study the skin corrosion potential of the test item was determined by means of the Human Skin Model Test (OECD 431). The relative absorbance value of the negative control the mean relative absorbance value
was 109.0% after 3 minutes exposure and 108.6% after 60 minutes exposure of the skin tissues to the test item. This value is above the threshold for corrosion of 50%. Therefore, the test item is not considered to possess corrosion potential.
Under the experimental conditions reported, the test substance is not corrosive to skin.

Executive summary:

This in vitro study was performed to assess the corrosive potential of the test substance by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

 

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. The test item (50 µL) was dispensed directly onto duplicate EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 =0.8 and =2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability <15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is =30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance values were not reduced compared with the negative control value, neither after 3 minutes exposure, nor after 1 hour exposure (109.0% and 108.6%, respectively). Both values did not touch the threshold for corrosivity, which is defined to be <50% after the 3 minutes exposure and <15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

 

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item was non corrosive to skin according to EU CLP and UN GHS.