Registration Dossier

Administrative data

Description of key information

The skin irritation potential of Fatty acids, lanolin, esters with cholesterol-low lanolin alcs. was assessed in vitro according to OECD Test Guideline 439. After a 65 minute exposure 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 (XcellR8 2017).

For the hydrolysis product Lanolin fatty acids an in vivo study no irreversible signs of irritation were observed and this substance is non-irritant to the skin (Davies 1968).

The substance was tested for eye irritation in the EpiOcular TM  Eye Irritation model according to OECD 492. 50 uL of the substance, positive control (methyl acetate) and negative control (H2O) was applied to the tissue for 6 hours, followed by a 25 minutes post-treatment immersion at 37°C, 5% CO2 and thereafter incubated for 18 hour post-dose at 37°C, 5% CO2 (XCellR8 2018). The substance did not interfere by colour or by direct MTT reduction. Tissue viability was 84.7% of negative controls, while validity criteria were met. The substance is concluded to be non-irritant.

In two eye irritation studies with Lanolin alcohols very limited signs of irritation to the iris were observed. The cornea and conjuctivae both produced some signs of irritation, however these values are not significant and are not enough to classify the material as an eye irritant (Davies 1968, Lewis 1977).

No signs of eye irritation were observed in a test with Lanolin alcohols in rabbits (Cade 1980)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
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
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.

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

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February 2018 to 01 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
- Tissue :Reconstructed human Cornea-like Epithelium (RhCE); keratincyte strain: 4F1188
- Model used: ‘EpiOcularTM irritation test ( MatTek Corporation EpiOcularTM EIT (OCL-200) Reconstituted Human Ocular Epithelium )
- Tissue batch number(s): (Lot # 27025)
- Delivery date: 25 February 2018
- Date of initiation of testing: 25 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 6h ± 15 minutes, followed by a 25 ± 2 minutes’ post-treatment immersion at 37°C, 5% CO2
- Temperature of post-treatment incubation: 18 hours ± 15 minutes’ post-treatment incubation at 37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: not indicated (according to MatTEek SOP)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: no data (according to MatTEek SOP)
- MTT concentration: not specified
- Incubation time: no data
- Spectrophotometer: BMG LabTech FluoStar Optima at 570 nm
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
Duration of treatment / exposure:
6h ± 15 minutes, followed by a 25 ± 2 minutes’ post-treatment immersion at 37°C, 5% CO2, 18 hours ± 15 minutes’ post-treatment incubation at 37°C, 5% CO2
Number of animals or in vitro replicates:
3 replicates
Details on study design:
tisues were wetted with 20 uL PBS before application of the substance or controls

negative control: sterile H2O
positive control: methyl acetate (neat)

number of replicates: 3
Irritation parameter:
other: viability (%)
Value:
84.7
Negative controls validity:
valid
Remarks:
OD570 1.902
Positive controls validity:
valid
Remarks:
viability 2.88%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Acceptance criteria passed

Acceptance criterion 1 The mean OD570 of the negative control(treated with PBS) tissues is > 0.8 and < 2.8
 
Acceptance criterion 2 The mean of the positive control relative percentage viability must be below 50% of the mean of the negative controls.
 
Acceptance criterion 3 The standard deviationof OD values between three replicates in each experimental condition must be ≤ 18%
 

Name

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

Sterile water

NC

1.902

0.090

100.000

4.75

4.745

No Category

Methyl Acetate

PC

0.055

0.049

2.882

2.569

89.140

No Prediction

Test substance-SO-(RB)

TA2

1.611

0.090

84.685

4.748

5.607

No Category

 

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not irritant when tested in the EpiOcular TM Eye Irritation Test
Executive summary:

The substance was tested for eye irritation in the EpiOcular TM  Eye Irritation model according to OECD 492. 50 uL of the substance, positive control (methyl acetate) and negative control (H2O) was applied to the tissue for 6 hours, followed by a 25 minutes post-treatment immersion at 37°C, 5% CO2 and thereafter incubated for 18 hour post-dose at 37°C, 5% CO2. 

The substance did not interfere by colour or by direct MTT reduction. Tissue viability was 84.7% of negative controls, while validity criteria were met. The substance is concluded to be non-irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the information available, the substance does not need to be classified for skin- and eye-irritation according to Regulation (EC) No 1272/2008 (CLP).