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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 439 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
dated 23 July 2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
27 April 2017

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
slightly brown flowable liquid
Details on test material:
Name LABDANUM ABSOLUTE
Batch no. 0117/1
Appearance slightly brown flowable liquid
CAS No. 89997-74-0
EINECS-No. 289-711-7
Production date Jan. 2017
Expiry date Jan. 2019
Storage Room Temperature (20 ± 5°C)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

In vitro test system

Test system:
human skin model
Source species:
other: reconstructed epidermises
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Justification for test system used:
Following the REACH bottom-up strategy described in the ECHA R.7a guidance, the SkinEthic™ Reconstructed Human Epidermis Model method was used to assess skin irritation.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ Reconstructed Human Epidermis Model, SkinEthic Laboratories, Lyon, France (RHE/S/17)
- Tissue batch number(s): 17-RHE-091
- Expiration date: September 11, 2017
- Date of testing: between 31 August 2017 and 07 September 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 minutes at room temperature
- Temperature of post-treatment incubation : 41 hours and 15 minutes at 37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: none, but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2.
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Wavelength: 570 nm
The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.2 (CV = 1.9%) (O.D. > 0.7)
- Barrier function: 4.8 h (4.0h < ET50 < 10.0 h)
- Morphology: 6 cells layers (> 4). Absence of significant histological abnormalities. Satisfactory (Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum)
- Contamination: absence of HIV1 & 2 antibodies, hepatitis C and antibodies, hepatitis C and antigen HBsp on blood; absence of mycoplasma on epidermal cells
- Reproducibility: All of the values for the negative and positive control groups fell within or very close to the historical ranges of the testing laboratory obtained in the previous year. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding approximately 50 mg of the test item applied on a nylon mesh to 1 mL of solution of MTT at 1 mg/mL. A yellow to brown solution was observed after 3 hours of incubation between 36.5°C and 37.8°C, 5% CO2.
> Therefore, there is no direct interaction between the test item and MTT.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is < 50% and in the absence of information on a skin corrosion test.
- The test substance is considered to be non-irritant to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µl
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5 % in distilled water
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
41 hours and 15 minutes post-incubation period at 37°C, 5% CO2
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Value:
3.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.6%
Other effects / acceptance of results:
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 3.6%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

- OTHER EFFECTS:
- Visible damage on test system: none but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse
- Direct-MTT reduction: none.
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.573 and 0.860). [The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.]
- Acceptance criteria met for positive control: yes, the positive control is classified as irritant
- Acceptance criteria met for variability between replicate measurements: yes, the SD values of the % viability are ≤ 18% (values between 0.1 and 16.7%).
- Range of historical values if different from the ones specified in the test guideline: The positive control OD were within the historical control range. The negative control OD of one of the replicate were below the historical control range. However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

Skin

OD

Mean OD / disc

(#)

Mean OD / product

Viability

%

Mean viability

%

Standard deviation

(SD)

Negative control

1

0.573

0.613

0.759

80.8

100.0

16.7

0.626

0.641

2

0.818

0.824

108.6

0.827

0.826

3

0.826

0.839

110.6

0.831

0.860

Positive control

1

0.012

0.013

0.012

1.7

1.6

0.1

0.013

0.013

2

0.011

0.011

1.4

0.011

0.011

3

0.013

0.012

1.8

0.011

0.011

Test item

1

0.040

0.037

0.028

4.9

3.6

1.1

0.036

0.036

2

0.020

0.020

2.6

0.020

0.020

3

0.027

0.026

3.4

0.026

0.026

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, in the absence of information on skin corrosion, the test substance has to be classified in Category 2 "Irritating to skin" or Category 1 "Corrosive" according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the SkinEthic reconstructed human epidermis model.

The test item was administered as supplied, at the dose of 16µl, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours 15 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues was 3.6%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

Concerning acceptability criteria:

- The negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.573 and 0.860).

- The SD values of the % viability are ≤ 18% (values between 0.1 and 16.7%).

- The positive control is classified as irritant.

- The 3 replicates of the test item gave concordant classification.

The quality criteria required for acceptance of results in the test were satisfied.

The positive control OD were within the historical control range. The negative control OD of one of the replicate were below the historical control range. However, this deviation is not considered to have affected the integrity of the study or the overall conclusion.

 

Under the experimental conditions of this study, in the absence of information on skin corrosion, the test substance has to be classified in Category 2 "Irritating to skin" or Category 1 "Corrosive" according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.