Registration Dossier

Administrative data

Description of key information

Skin:
The test substance did not show a skin corrosive or irritating property in the EpiDerm in vitro assay.
Eye:
The test substance was shown to have no eye irritating and eye damaging potential in the BCOP Test and in the EpiOcular Eye Irritation test.

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 irritation

In order to assess the potential for corrosive activity and skin irritation of the test substance, two in vitro assays using the EpiDermTM model were performed, one according to OECD Guideline 431 and the other according to OECD Guideline 439.

For testing of a corrosive property two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator) and test group (test material, negative control and positive control) were used. 25 μL of the solid test material in 25 μL de-ionized water was applied. Control tissues were concurrently treated with 50 μL of de-ionized water (negative control) or with 50 μL of 8 N potassium hydroxide (positive control). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm of the extracts was determined spectrophotometrically. As a result, the mean tissue viability after 3 min exposure was 100 % (compared to the negative control) and 113 % after 1 hour exposure. Therefore, the substance was considered to be non corrosive.

For testing of an irritating property of the test substance, three tissues each were treated with the test substance, the positive control substance (30 μL of 5 % SDS) and the negative control substance (30 µL PBS), respectively. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. The mean tissue viability was determined to be 99 % compared to the negative control and therefore the substance was considered to have no irritating potential to the skin.

Eye irritation

To assess the potential of the test substance to cause eye irritation and severe eye damage, two in vitro assays were conducted: a Bovine Corneal Opacity and Permeability Test (BCOP Test) and an EpiOcular Eye Irritation Test.

The BCOP Test was conducted according to OECD Guideline 437. A single topical application of 750 μL of a 20 % test substance preparation to the epithelial surface of isolated bovine corneas was performed.Three corneas were treated with the test substance for an exposure period of 4 hours. In addition to the test substance a negative control (de-ionized water) and a positive control (20 % imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance. The mean IVIS score of the test substance-treated eyes was caclulated to be 1.6 and therefore well below the mean positive control score of 101.2, the mean IVIS score of the negative control eyes was calculated to be 7.9. In addition H&E-stained cross sections of the corneas were evaluated for the irritation potential of the test substance. No histopathological findings were made. Based on these results it was concluded that the test substance did not cause severe eye damage.

The EpiOcular test was performed administering a single topical application of 50 μL bulk volume (about 49 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcularTM eye irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 112 %. Based on the observed results it was concluded that the test substance does not show an eye irritating potential.


Justification for selection of skin irritation / corrosion endpoint:
Two GLP and Guideline studies are available for this endpoint which were used in a weight of evidence apporach.

Justification for selection of eye irritation endpoint:
Two GLP and Guideline studies are available for this endpoint which were used in a weight of evidence apporach.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin or eye irritation under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC 605/2014.