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Administrative data

Description of key information

Based on the results of a study according to OECD TG 431 the test item is identified as skin corrosive.

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
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)
Version / remarks:
14 June 2019
Deviations:
no
Qualifier:
according to
Guideline:
other: B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”
Version / remarks:
30th May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
artificial membrane barrier model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin, EPISKIN SNC Lyon, France
- Tissue batch number: 20-EKIN-008

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL PBS 1 x solution
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in saline buffer
- Incubation time: 3 hours (± 15 min) at 37±1°C in an incubator with 5±1 % CO2, protected from light, ≥95 % humidified atmosphere.
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Morphology: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum
- Contamination: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: Water-killed epidermis
Place the living epidermis in a 12 well plate with 2 mL of distilled water (replacing the culture medium).Incubate at 37 °C, 5 % CO2, ≥ 95 % humidified atmosphere for 48 hrs +/- 1 hour. At the end of the incubation, discard the water. Keep dead epidermis frozen (dry) in freezer at -15 °C to -30 °C (killed epidermis can be stored and used up to 6 months). Before use, the killed tissues are de-frozen at room temperature (approx. 1 hour in 2 mL of maintenance medium).
- N. of replicates: 2
- Method of calculation used: As indictaed in guideline.

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

PREDICTION MODEL / DECISION CRITERIA
- see table 1
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 0.9%

POSITIVE CONTROL
- Amount(s) applied: 50 µL
Duration of treatment / exposure:
exposure times of 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment (4 hours exposure)
Value:
24
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment (1 hours exposure)
Value:
57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment (3 min exposure)
Value:
40
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The test item interacted with the MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 20.67 %, 18.60 % and 39.02 % at the 4h, 1h and 3 min exposure respectively. As the NSMTT were below 50 % in each case the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: The test item is colourless, furthermore showed no ability to become coloured in contact with water. Therefore, considered not to be able to significantly stain the tissues and lead to false estimate of viability. Additional controls and data calculations were not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency in a separate study (study no.: 392-431-4224) using the twelve Proficiency Chemicals according to OECD Test Guideline No. 431.

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
Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
Based on the results of the study according to OECD TG 431 the test item can be classified as Corrosive: a combination of optional Subcategories 1B-and-1C.
Executive summary:

A study according OECD TG 431 was conducted to predict the corrosion potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431 (adopted 14 June 2019).

Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item treated tissue viabilities were below 35 % of the mean negative control value after 4 hours exposure and above 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue relative viabilities were 24 % at 4 hours, 57 % at 1 hour and 40 % at 3 minutes of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals skin corrosion potential after 4 hours and no skin corrosion potential after 1 hour and 3 min of exposure under the utilised testing conditions. Based on the results above the test item can be classified as Corrosive: a combination of optional Subcategories 1B-and-1C.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study according OECD TG 431 was conducted to predict the corrosion potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431 (adopted 14 June 2019).

Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item treated tissue viabilities were below 35 % of the mean negative control value after 4 hours exposure and above 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue relative viabilities were 24 % at 4 hours, 57 % at 1 hour and 40 % at 3 minutes of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals skin corrosion potential after 4 hours and no skin corrosion potential after 1 hour and 3 min of exposure under the utilised testing conditions. Based on the results above the test item can be classified as Corrosive: a combination of optional Subcategories 1B-and-1C.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for skin corrosion Cat.1B (H314) and eye damage Cat.1 (H318) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.