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Administrative data

Description of key information

not skin irritating

not eye irritating

Key value for chemical safety assessment

Skin irritation / corrosion

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

Eye irritation

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN CORROSION/ IRRITATION

The potential of the test item to be irritant to the skin was investigated through anin vitroskin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline 439 (2015). The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A dark brown solution was noted at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A dark brown solution was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 2.855, indicating that the test item has a potential interfering ability. Based on these results, additional controls were added in theMain Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 ± 2 mg/epidermis unit, each measuring 0.38cm2 (treatment level: 53mg/cm2).

Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues withoutMTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues.

Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.

In the Main Assay, the negative control gave the expected baseline value and variability, in agreement with the guideline indications.

The positive control caused the expected cell death and variability. Based on the stated criteria, the assay was regarded as valid.

The NSMTT value was −1%, while the NSCliving value was 1%, thus only the OD-blank background subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 100 %, when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 8.5.

Based on the results obtained, the test item is not classified as irritant to the skin according to the CLP Regulation EC no. 1272/2008.

SERIOUS EYE DAMAGE/EYE IRRITATION

The eye hazard potential of the substance is evaluatedin vitroin a Reconstructed human Cornea-like Epithelium (RhCE) model according to the OECD 492 Guideline. Before the main test the test item was assessed for its ability to directly reduce the vital dye MTT and also whether it has colour interference.As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement.Therefore, an additional test for intensely coloured test items was performed.

In the main test, the test itemwasapplied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control.

The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 44.3 % (< 50%). Validity criteria were met and therefore the test is considered as valid.

After treatment with the test item, the mean value of relative tissue viability was reduced to 72.9 %. This value is above the threshold for eye irritation potential (≤ 60 %).

Under the conditions of the test, the test material is considered non-eye irritant.

Justification for classification or non-classification

SKIN CORROSION/ IRRITATION

According to the CLP Regulation (EC) no. 1272/2008, the Skin Irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours. In vitro alternatives that have been validated and accepted may also be used to help make classification decisions. Table R.7.2-2 in the Guidance on IR&CSA lists the status of validation and regulatory acceptance for in vitro test methods for skin corrosion and skin irritation. The OECD has adopted an in vitro skin irritation test guideline i.e. OECD TG 439 (TM B. 46) that currently contains four test methods including the EpiSkinTM. This test method allows distinction between irritants (CLP Cat 1/Cat 2) and substances not classified. Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Based on the results obtained in the test performed on the substance, the mean cell viability after the blank subtraction was 100 % when compared to the negative control. Thus, no classification for the skin irritation is warranted according to the CLP Regulation (EC) no. 1272/2008.

SERIOUS EYE DAMAGE/EYE IRRITATION

According to the CLP Regulation (EC) no. 1272/2008, Serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application. The classification system for substances involves a tiered testing and evaluation scheme, combining pre-existing information on serious ocular tissue damage and on eye irritation as well as considerations on (Q)SAR and the output of validated in vitro tests in order to avoid unnecessary animal testing.

The OECD has at present adopted five in vitro test guidelines for assessing eye hazard potential. These tests are recommended for use as part of a tiered-testing strategy for regulatory classification and labelling.

Test method OECD TG 492 – Reconstructed human Cornea-like Epithelium Test Method (RhCE) is an in vitro assay that may be used to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage. The only in vitro test method currently covered by this Test Guideline is the EpiOcular™ Eye Irritation Test (EIT). The percentage tissue viability cut-off value for identifying test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) is > 60 %.

Based on the results obtained in the RhCE test performed on the substance, the mean value of tissue viability was 72.9 %. This value is above the threshold for eye irritation potential (60 %). Thus, the substance is not considered as eye irritant according to the CLP Regulation (EC) no. 1272/2008.

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