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Description of key information

Skin corrosion in vitro: The test item was considered to be non-corrosive to skin because cell viability was determined to be 103.3 % after 3 minutes and 104.4 % after 60 minutes (OECD 431; EpiDerm Human Skin Model).

 

Skin irritation in vitro: The test item was considered to be non-irritant to skin because the relative mean viability of the test item treated tissues was determined to be 73.3 % after the 15-minute exposure period and 42-hours post-exposure incubation (OECD 439; EPISKIN reconstructed human epidermis model).

 

Eye damage/irritation in vitro: The test item was found to be non-hazardous to the eye because the in vitro irritancy score (IVIS) was 2.1 in the BCOP assay (OECD 437 and EU Method B.47).

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 in vitro

The key study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was 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 and cytotoxicity is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium 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. Negative and positive control groups were treated for each exposure period. 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 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 5.8 % (positive control) and 103.3 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.6 % (positive control) and 104.4 % (test item).

Skin irritation in vitro

The key study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (adopted 28 July 2015) and Method B.46 in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINT reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 73.3 % after the 15-minute exposure period 42 hour post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

Skin irritation in vivo

Skin irritation does not need to be investigated in vertebrate animals because a conclusive classification decision resulted from in vitro data. The test material was demonstrated to be non-hazardous using validated in vitro methods (cell viability ≥ 50 % after 3 minutes and ≥ 15 % after 60 minutes using OECD 431 and percentage tissue viability after exposure and post-treatment incubation > 50 % using OECD 439).

Eye irritation in vitro

The key study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). In Vitro Irritancy Scores were reported as 2.1 for the test item, 0.8 for the negative control and 52.3 for the positive control.

 

Eye irritation in vivo

Eye irritation potential does not need to be investigated in vertebrate animals because a conclusive classification decision resulted from in vitro data. The test material was demonstrated to be non-hazardous (In vitro irritancy score ≤ 3) using the Bovine Corneal Opacity and Permeability (BCOP) assay.

Justification for classification or non-classification

Skin corrosion:The test item was considered to be non-corrosive to skin because cell viability was determined by OECD 431 (28 July 2015) to be 50 % after 3 minutes and 15 % after 60 minutes using the EpiDerm Human Skin Model. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Skin irritation:The test item was determined by OECD 439 (28 July 2015) to be non-irritant to skin because percentage tissue viability after exposure and post-treatment incubation was found to be > 50 % using the EPISKIN reconstructed human epidermis model. Classification for skin irritation in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Eye damage/irritation:The in vitro irritancy score (IVIS) was determined to be ≤ 3 for the test item in the BCOP assay and, in accordance with OECD 437 (26 July 2013), no classification is required under the terms of GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments.

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