Fatty acids, lanolin, esters with cholesterol-low lanolin alcs.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2017 to 22 September 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, lanolin, esters with cholesterol-low lanolin alcs.

EC Number:

295-374-7

EC Name:

Fatty acids, lanolin, esters with cholesterol-low lanolin alcs.

Cas Number:

92044-94-5

Molecular formula:

not applicable for UVCB

IUPAC Name:

Fatty acids, lanolin, esters with cholesterol-low lanolin alcs.

Test material form:

solid - liquid: suspension

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed Human Epidermis

Cell source:

other: MatTek kit

Justification for test system used:

Description EpiDermTM (EPI-200) Reconstructed Human Epidermis
Lot Number 25842
Date of Receipt 19th Sep 2017
Morphology EPI-200 LOT#: 25842
Histological examination demonstrates human epidermis-like structure: including multiple layers (at least 4) of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) which are present under multilayered stratum corneum: 9 layers--> - PASS
Tissue thickness: 95.5 µm --> Acceptance range: >70 µm and <130 µm) - PASS
Tissue Viability: Optical Density (O.D.) values: 1.39 ± 0.046 (mean and SD of MTT value of 3 tissues exposed to H2O) -->Acceptance range: [1.0 - 3.0]- PASS
Skin Barrier Function: ET 50% of tissues exposed to 100 µL Triton X-100 1% (n=3): 7.12 h -->Acceptance range: [4.77h- 8.72h] - PASS
Sterility: No evidence of contamination during long term antibiotic and antimycotic free culture: No contamination reported --> - PASS

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue batch number(s): 25842
- Delivery date: 19 September 2017
- Date of initiation of testing: 25 September

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 31 minutes at room temperature and 34 minutes at 37°C, 5% CO2, ≥95% RH
- Temperature of post-treatment incubation: 42 ± 4h post-treatment incubation

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsing (no details)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: no data
- Incubation time:no data
- Spectrophotometer: BMG LabTech FluoStar Optima (calibrated on 24 July 2017)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: not indicated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: based on blanks obtained during 66 historical runs (no data provided)

NUMBER OF REPLICATE TISSUES: 3/treatment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
•A test item is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.
•A test item may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mgTEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg neat


NEGATIVE CONTROL:Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) neat

POSITIVE CONTROL: 5% Sodium dodecyl sulphate (SDS)
- Concentration (if solution): 5% in water

Duration of treatment / exposure:

31 minutes at room temperature and 34 minutes at 37°C, 5% CO2, ≥95% RH

Duration of post-treatment incubation (if applicable):

42 ± 4h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

97.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Prior to the study, the required compatibility checks confirmed that the test item did not interfere with MTT and no water colouration was observed.

Acceptance criterion 1 The mean OD570 of the negative control (treated with DPBS) tissues must be ≥ 0.8 and ≤ 2.8  1.514 PASS
Acceptance criterion 2 The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls.  5.097 PASS
Acceptance criterion 3 The standard deviation of viability percentages for triplicate skin models in each experimental condition must be < 18%  NC: 5.243; PC: 1.176; TA2: 12.886 PASS
Acceptance criterion 4 The mean OD of the 6 wells containing extraction solvent alone (blanks) must be ≤ 0.1.  0.162 FAIL

blank controls:
Optical Density (OD) values obtained with blanks were higher than 0.1 (0.162) causing a deviation from the acceptance criteria. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs), therefore this is not considered to be an issue in the interpretation of this study data.

Any other information on results incl. tables

Viability measurements after 65 min of application and 42h (± 4h) post-incubation of test and reference items and controls. 

Condition	Tissue #	Raw data		Blank corrected data		Mean OD	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	1.781	1.753	1.619	1.591	1.605	106.022
	Tissue 2	1.657	1.587	1.495	1.425	1.460	96.444
	Tissue 3	1.663	1.614	1.501	1.452	1.477	97.534
PC	Tissue 1	0.268	0.251	0.106	0.089	0.098	6.451
	Tissue 2	0.224	0.231	0.062	0.069	0.066	4.337
	Tissue 3	0.226	0.234	0.064	0.072	0.068	4.502
TA2	Tissue 1	1.481	1.453	1.319	1.291	1.305	86.207
	Tissue 2	1.657	1.552	1.495	1.390	1.443	95.288
	Tissue 3	1.859	1.845	1.697	1.683	1.690	111.636

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA2: test substance

Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
NC	1.514	0.079	100.000	5.243	5.243	Non-Irritant
PC	0.077	0.018	5.097	1.176	23.064	Irritant
TA2	1.479	0.195	97.710	12.886	13.188	Non-Irritant

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of the study the substance is considered not irritating to the skin.

Executive summary:

The skin irritation potential of the substance was assessed in vitro according to OECD Test Guideline 439. After exposure during 31 minutes at room temperature and 34 minutes at 37°C, 5% CO2, ≥95% RH on the surface of the EpiDermTM reconstructed human epidermis and a 42 ± 4h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control. The percentage of viability obtained was 97.710 % and therefore the substance is considered as non-irritant to the skin