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Description of key information

The in vitro skin corrosion of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 431. The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.

After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density

(OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The Relative mean tissue viability calculated as a percentage of the negative control was 6.5% after the 60-minute exposure and 81.9% after the 3-minute exposure. The test item met the criteria for classification as corrosive to the skin (Skin Corr 1B) according to Regulation (EC) No.1272/2008.

 

In accordance with Annex VII of REACH, the substance being classified as corrosive to the skin, the eye irritation and skin irritation studies do not need to be conducted.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 March 2018 to 20 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
see 'Principles of method if other than guideline'
Principles of method if other than guideline:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 25896
- Delivery date: 17 April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
Duplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
81.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
6.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: See 'Any other information on results incl. tables'
- Colour interference with MTT: None

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

 An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes. The results generated from the freeze-killed tissues are presented below.

 

3 minutes exposure:

Mean of test item treated killed tissues (tkt) = 0.118 OD570

Mean of untreated killed tissues (ukt) = 0.120 OD570

The direct reduction by the test item relative to the negative control value:

(0.118 (tkt)–0.120 (ukt)) / 2.080 (mean of negative control) = 0.0%

 

60 minutes exposure:

Mean of test item treated killed tissues (tkt) = 0.119 OD570

Mean of untreated killed tissues (ukt) = 0.121 OD570

The direct reduction by the test item relative to the negative control value:

(0.119 (tkt)–0.121 (ukt)) / 2.213 (mean of negative control) = 0.0%

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
The Relative mean tissue viability calculated as a percentage of the negative control was 6.5% after the 60-minute exposure and 81.9% after the 3-minute exposure. The test item met the criteria for classification as corrosive to the skin (Skin Corr 1B) according to Regulation (EC) No.1272/2008.
Executive summary:

The in vitro skin corrosion of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 431. The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.

After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density

(OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The Relative mean tissue viability calculated as a percentage of the negative control was 6.5% after the 60-minute exposure and 81.9% after the 3-minute exposure. The test item met the criteria for classification as corrosive to the skin (Skin Corr 1B) according to Regulation (EC) No.1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Additional information

Justification for classification or non-classification

In accordance with Regulation (EC) No.1272/2008, Trans-hex-2-en-1-ol was classified as Skin Corr 1A; H314 and Eye Dam 1; H318.