Registration Dossier

Administrative data

Description of key information

Skin irritation: Not irritating to the skin

Eye irritation: the test material showed effects on the cornea of the bovine eye in vitro and is considered to be irritating to the eye.

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

Skin irritation

 

In a key Guideline (OECD 439) in vitro skin irritation study, the potential of the test material to be irritant to the skin was investigated using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The test material, 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.

 

A preliminary test was carried out to evaluate the compatibility of the test material with the test system. In the first step, the test material was assayed for to evaluate its ability to reduce MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS N. 298-93-1). A grey purple solution was observed at the end of the incubation period, indicating that the test material could directly interact with MTT. In the second step, the test material was assayed for the ability of colouring water and spectral analysis of the test material in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. A colourless solution was observed, however the value obtained for the Optical Density (OD) was 0.217, indicating that the test material has a potential interfering ability. Based on these results, additional controls were added in the Main Assay.

 

In the Main Assay, the test material was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2(treatment level: 53 mg/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 20μL/epidermis unit. To verify if the test material results had to be corrected, the non-specific colour (NSCliving) was evaluated using two alive treated tissues without MTT 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 material was 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.

 

Data presented for the alive tissues treated with the test material were obtained in a repeated experiment (second run). In the first run, probably due to a microbial contamination in two epidermis units, unacceptable variability within replicate tissues stained with MTT was observed. Since the non-specific colour (NSCliving) control needs to be performed concurrently to the testing of the coloured test chemical, this control was also repeated. In each run, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

 

The positive control caused expected cell death (mean viability ≤40%) and variability (SD of % viability lower than 18). Based on the stated criteria, the assay was regarded as valid. Following treatment of alive tissue without MTT, no relevant colouring ability of the test material was noted; no relevant interaction was recorded between the test material and MTT. Based on these results, only the OD-blank background subtraction was performed. In the second run, the test material did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 98%, when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 4.5 (lower than 18, as stated in the Study Protocol).

 

Based on the results observed, the test material is considered as ''not classified” or the criteria for “classification“ are not satisfied.

 

Eye Irritation

 

A key Guideline (OECD 437) in vitro eye irritation/corrosion study was performed to assess the potential of the test material to cause corneal damage by quantitatively measuring changes in opacity and permeability in bovine cornea post exposure.

 

The test material was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 hour and whose opacity had been measured. The test material incubated on the cornea for 3 hours and 55 minutes at 32 ± 1°C. After removal of the test material, opacity and permeability values were measured. HBSS-solution was used as the negative control while 20% Imidazole solution was used as the positive control in this study.

 

The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) was 2.79. The positive control induced serious eye damage on the cornea and was within two standard deviations of the historical mean. The calculated IVIS for the positive control was 115.24. The calculated IVIS for the test material was 27.41.

 

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in a UN GHS Category for eye damage. Therefore, under the conditions of this study, the test material showed effects on the cornea of the bovine eye in vitro but does not meet the GHS classification criteria for eye damage.

Respiratory Irritation

No data available for assessment

Justification for classification or non-classification

Not classified for skin irritation according to CLP Regulation (EC 1272/2008), section 3.2 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Classified as Category 2 (H319: causes serious eye irritation) according to CLP Regulation (EC 1272/2008), section 3.3 but does not meet the GHS classification criteria for eye damage.