Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Apr - 30 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, LAndwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

EC Number:

248-813-1

EC Name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Cas Number:

28065-97-6

Molecular formula:

C34H69NO2 C36H67NO2 C36H69NO2 C36H71NO2 C36H73NO2

IUPAC Name:

oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ tissues

Source strain:

other: The cells used to produce EpiDerm™ tissues are derived from tissue obtained by MatTek Corporatio (keratinocyte strain: 00267).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues
- Tissue batch number(s): 25825
- Delivery date: 27 June 2017
- Date of initiation of testing: 27 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 min at 37 ± 1.5 °C and 25 min at room temperature
- Temperature of post-treatment incubation: 41 h at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissue viability was assessed by undertaking a MTT quality check test and resulted in a an OD (540 - 570 nm) value of 1.775 ± 0.121
- 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 5.69 h
- Morphology: appropriate formation of the epidermal barrier, the presence of a funtional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers were determined
- Contamination: the cells were screened for potential biological contaminants. A potential contamination with HIV-1 virus, Hepatitis B virus, Hepatitis C virus and Bacteria, yeast and other fungi was not detected

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test item did not stain water, and additional test with viable tissues was not necessary to be performed. Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to have an irritation potential to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

ACCEPTABILITY CRITERIA
1) The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.
2) An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
3) The rel. standard deviation (SD) of 3 identical replicates should be ≤ 18%.
OD values should not be below historically established boundaries.

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: 5% in deionised water

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

41 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:

60 min exposure

Value:

25.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: MTT was not reduced by the test substance.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD values of 3 wells of the three tissues were 1.722, 1.776 and 1.625, respectively, and thus in the range of ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: The mean tissue viability was 3.3% and thus meeting the acceptance criterion.
- Acceptance criteria met for variability between replicate measurements: The relative SD values of the three tissues treated with the negative and positive control and the test substance were 4.6, 0.6 and 10.3%, respectively, and thus ≤ 18%.

Any other information on results incl. tables

Table 1: Results

Dose Group	Tissue No.	Absor- bance 570 nm Well 1	Absor- bance 570 nm Well 2	Absor- bance 570 nm Well 3	Mean Absor- bance of 3 Wells	Mean- Absor- bance of three Wells blank corrected	Mean Absor- bance of 3 Tissues	Stand. Deviation	Rel. Absor- bance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]**	Mean Rel. Absorbance [%]***
Blank		0.038	0.038	0.037	0.037	0.000
Negative Control	1	1.708	1.718	1.740	1.722	1.684	1.670	0.076	100.8	4.6	100.0
	2	1.804	1.757	1.767	1.776	1.738			104.1
	3	1.659	1.606	1.611	1.625	1.588			95.1
Positive Control	1	0.090	0.095	0.094	0.093	0.055	0.055	0.000	3.3	0.6	3.3
	2	0.093	0.093	0.091	0.092	0.055			3.3
	3	0.092	0.093	0.093	0.093	0.055			3.3
Test Substance	1	0.413	0.408	0.409	0.410	0.373	0.422	0.043	22.3	10.3	25.2
	2	0.482	0.468	0.471	0.474	0.436			26.1
	3	0.494	0.484	0.501	0.493	0.456			27.3

* Relative absorbance [rounded values]: 100 x (absorbance (test substance or positive control or negative control)) / (mean absorbance negative control)

** Relative standard deviation [rounded values]: 100 x standard deviation / mean absorbance

*** Mean relative absorbance [rounded values]: 100 x (mean absorbance (test substance or positive control or negative control)) / (mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: Skin Irrit. 2, H315 according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the EpiDerm™ model. The result does not allow for the classification of the test substance as irritant or corrosive and therefore further evaluation and/or data generation is required. Based on the results of the available in vitro skin corrosion test according to OECD 435 the test substance did not show corrosive properties towards the skin in the in vitro Membrane Barrier Test for Skin Corrosion using Corrositex™. Therefore the test substance can be considered as a skin irritant.

CLP: Skin Irrit. 2, H315