Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

adopted 18 Jun. 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Comission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, EU method B.46 'In vitro skin irritation: reconstructed human epidermis model test'

Version / remarks:

adopted 06. Jul. 2012

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Mainz, Germany

Test material

Test material information

1

Reference substance name:

6-[(9Z)-octadec-9-enoyloxy]hexyl (9Z)-octadec-9-enoate; 6-hydroxyhexyl (9Z)-octadec-9-enoate

EC Number:

947-912-3

Molecular formula:

Not applicable for UVCB

IUPAC Name:

6-[(9Z)-octadec-9-enoyloxy]hexyl (9Z)-octadec-9-enoate; 6-hydroxyhexyl (9Z)-octadec-9-enoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM (EPI-200-SIT)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200-SIT) (MatTek In Vitro Life Science Laboratories, Bratislava)
- Tissue batch number(s): 30808
- Delivery date: 23. Jul. 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: M icrotiter plate photometer Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.841 ± 0.032 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.26 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce EpiDerm™ tissue are screened for potential biological contaminants. Tests for the following potential biological contaminant were performed: HIV-1 virus (Oligonucleotide-direct amplification), Hepatitis B virus (Oligonucleotide-direct amplification), Hepatitis C virus (Oligonucleotide-direct amplification), Bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture); no contaminations detected.
- Reproducibility: given

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Direct MTT reduction by the test item had not taken place and no data correction was necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive / irritant to skin if the tissue viability after 1 h is equal or less than 50%
- The test substance is considered to be non-irritant to skin if the tissue viability after 1 h exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

35 min at 37°C and 25 min at RT

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

1 h exposure

Value:

106.4

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:

- OTHER EFFECTS:
- Direct-MTT reduction: Direct MTT reduction by the test item had not taken place and no data correction was necessary.
- Colour interference with MTT: The solution was colourless, therefore no binding capacity had to be tested.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.429 and 1.658).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour was ≤ 20% compared to the negative control (3.1%).
- Acceptance criteria met for variability between replicate measurements: The SD of mean viability of the tissue replicates is ≤ 18% (values between 0.1% and 9.8%).
- Range of historical values if different from the ones specified in the test guideline: The mean OD of the tissue replicates treated with the negative control of all experiments up to 03. Jul. 2019 was 0.476 - 2.471 (mean: 1.787, SD 0.317).

Any other information on results incl. tables

Table 1: MTT assay after 60 min exposure

	Negative control			Positive control			Test item
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	1.585	1.702	1.443	0.080	0.081	0.082	1.523	1.770	1.752
	1.586	1.682	1.482	0.080	0.081	0.084	1.519	1.760	1.749
OD570(mean-blank)	1.552	1.658	1.429	0.046	0.047	0.049	1.487	1.731	1.717
OD570(mean of 3 tissues)	1.546			0.047			1.645
Viability (%)	100			3.1			106.4
SD	-			0.1			8.9

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008