Hexa-1,5-diene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

EpiDerm Human Reconstructed Epidermis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 8 January 2018 Experimental completion date: 12 January 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Version adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC B.40 BIS

Version / remarks:

EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hexa-1,5-diene

EC Number:

209-754-7

EC Name:

Hexa-1,5-diene

Cas Number:

592-42-7

Molecular formula:

C6H10

IUPAC Name:

hexa-1,5-diene

Specific details on test material used for the study:

1,5-hexadiene. 99.4% purity.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch:1 June 2018
- Purity test date: 26 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Analyses conducted to support the information cited in the certificate of analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality system.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human donor skin

Vehicle:

unchanged (no vehicle)

Details on test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis (provided by MatTek Corporation, Ashland MA, USA). It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.ç

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 27667
- Delivery date: 10 January 2018
- Date of initiation of testing: 8 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC for the 1-hour exposure; room temperature for the 3-minute exposure
- Temperature of post-treatment incubation (if applicable): N/A

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 wash with PBS, no volume specified
- Observable damage in the tissue due to washing: N/A

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL undiluted test item.
50µL Milli-Q water for the negative control.
50 µL 8N KOH for the positive control.

Duration of treatment / exposure:

2 tissues exposed for 3 minutes.
2 tissues exposed for 1 hour.

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

4 tissues per test item (2 tissues per exposure time) together with 1 negative and 1 positive control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

> 90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ¿0.8 and upper acceptance limit equal to or below 2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 4.7%. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was equal to or below 21%, indicating that the test system functioned properly.

Any other information on results incl. tables

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with 1,5-hexadiene compared to the negative control tissues was 91% and 109% respectively.

Mean tissue viability in the in vitro skin corrosion test with 1,5 -hexadiene:

	3 -min application viability (% of control)	1 -hour application viability (% of control)
Negative control	100	100
1,5 -hexadiene	91	109
Positive control	6.7	4.7

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

1,5-hexadiene is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate 1,5-hexadiene for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of 1,5-hexadiene was tested through topical application for 3 minutes and 1 hour. The study procedures were based on the OECD 431 guideline. Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colourless liquid. 1,5-hexadiene was applied undiluted (50 ¿L) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 4.7% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ¿0.8 and upper acceptance limit equal to or below 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was equal to or below 21%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 91% and 109%, respectively. Because the mean relative tissue viability for 1,5-hexadiene was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment 1,5-hexadiene is considered to be not corrosive. In conclusion, 1,5-hexadiene is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the study report.