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Administrative data

Description of key information

Eye-irritant

Non skin irritant

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:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The skin and eye irritation of the substance were evaluated in vitro.

The skin irritation of the substance was evaluated in-vitro in the SkinEthic 3D reconstructed artificial human skin model, according to the OECD Guideline 439. The test material was applied on each tissue in three replicates. After 42 minutes of exposure, the substance was removed and the tissue was incubated at 37 °C, 5 % CO2 for 42 hours. At the end of the exposure the MTT assay was performed to evaluate the tissue cell viability. Negative controls (phosphate buffer) and positive (5 % SLS) controls run in parallel. The tissue cell viability was 117.21 % for the substance, 1.81 % for positive control and 100.00 for negative control.

The tissue viability is more than 50 % therefore the substance is considered as a non-skin irritant.

The eye irritation potential was evaluated according to the HET-CAM assay and the NRU assay. HET-CAM is a validated assay which is used in combination with NRU Assay in order to classify severe ocular irritants with sufficient reliability (ECVAM DB-ALM INVITTOX Protocol No 47). Τhe NRU asasay is one of the in vitro alternative methods developed to refine and eventually to replace the Draize rabbit eye test for the classification and labelling of eye irritants.

The corrosion potential of the substance was evaluated in the in-vitro HET CAM test according to Luepke, N.P.(1985), DB-ALM Protocol No 47 and 96 (ECVAM). Six fertilised eggs from White Leghorn hens, were incubated for eight days at 37.8 ± 1 °C, 50 -60 % relative humidity and were then exposed to the substance for 300 seconds. During the exposure period the chorioallantoic membrane of the egg was evaluated for the presence of lysis of blood vessels, coagulation and haemorrhage. The irritation score (IS) was then evaluated taking in conideration the appearance time of each reaction. The irritation score of the substance was calculated to be 0.25 ± 0.18.

The eye irritation potential of the substance was quantitavely assessed by the cytotoxicity assay by using the NRU (Neutral Red Uptake) assay. Fibroblasts Balb 3T3 clone 31 were exposed to the substance at eight concentrations ranging from 2000 μg/ml to 15.6 μg/ml, for 24 hours. At the end of the incubation period the medium was removed and the cells were washed with PBS and fresh culture medium and Neutral Red dye was added to the cells. The cells were incubated for 3 hours at 37 °C and 5 % CO2, after which the NR-medium was removed, the cells were washed with PBS to solubilise the NR captured by the cells. The absorbance of neutral red was measured at 540 nm. Negative controls (corn oil and culture medium) and positive control (SDS) run in parallel. The cell viability and the NR50 were calculated. The NR50 of the sample was calculated to be > 1000 μg/ml corresponding to a MMAS < 25. The substance is considered to have a no/mild irritation eye potential.

Justification for classification or non-classification

Skin irritation

The substance is not classified as a skin irritant according to the CLP Regulation (EC) No.1272/2008 as the tissue viability obtained in the study is above the threshold of viability indicated in the OECD 439 in order to be considered as a skin irritant.

Eye irritation

According to the DB-ALM Protocol no 100 (June, 2017): Neutral Red Uptake (NRU) Bioassay using BALB/c 3T3 cells, determination of NRU in 3T3 cells has been proposed as an in vitro alternative to Draize rabbit eye irritancy test. According to the DB-ALM Method Summary no 3 (February, 2013): Neutral Red Uptake (NRU), in the context of occular irritation, the assay is generally applied as part of a test battery or as adjunct assay in a tiered approach to assess substances that are ocular irritants, e.g. in combination with other cell based assays or in combination with an organotypic in vitro assay, for instance HET-CAM or the IRE test. The NRU assay has been originally employed as a potential replacement of the Draize rabbit eye test for the classification and labelling of eye irritants.

According to the Guidance on the Application of the CLP Criteria (ECHA, 2017) the Hen's Egg Test on Chorioallantoic Membrane (HET-CAM) test is recommended for use as part of a tiered testing strategy for regulatory classification and labelling (e.g. Top-Down Approach). A substance can be considered as causing serious eye damage (Category 1) based on positive results in the ICE test, the BCOP test, the FL test, the STE test, CM test IRE test or the HET-CAM test.

The substance is found to be non-eye irritant in both studies. However, a negative result in these studies cannot be used for the non-classification of the substance according to the CLP Regulation (EC) No. 1272/2008. The available results exclude the serious eye damage potential of the substance, however, following a precautionary approach, the substance is classified as an Eye Irritant 2 (H319).

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