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Administrative data

Description of key information

Not skin irritant

Eye irritant

Key value for chemical safety assessment

Additional information

Skin irritation/corrosion

EpiSkinTMSM test has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2,≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2,≥95 % humidified atmosphere andprotected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item has an intrinsic colour (purple), therefore two additional test item treated tissues were used for the non-specific OD evaluation (NSCliving).

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (purple). To avoid a possible double correction [TODTT (MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

In thisin vitroskin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean true tissue viability: 124.0%). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18.The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model (OECD 439), indicated that the test item reveals no skin irritation potential under the utilised testing conditions.

Eye irritation/corrosion

The purpose of the first study, the Isolated Chicken Eye Test (ICET), was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes, according to the OECD guideline 438. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The Imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control.

Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µl saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 ml saline solution at ambient temperature and this procedure was repeated for each eye.

Adherence of the test item and the positive control Imidazole was observed on the cornea surfaces at 240 min after the post-treatment rinse.

In this ICET, test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xII, 2xIII.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

Overall based on the results obtained in this test “no prediction can be made”.

The purpose of the second study was to determine the acute ocular irritation potential of the test item Acid Red 143 on three-dimensional RhCE tissue in the EpiOcular™ model in vitro,according to the OECD guideline 492.

Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2°C in an incubator with 5 ± 1% CO2, ≥95% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2°C in an incubator with 5 ± 1% CO2protected from light, ≥95% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (purple), therefore two additional test item treated tissues were used for the non-specific OD (Optical Density) evaluation (NSCliving).

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (purple). To avoid a possible double correction [TODTT*(MTT and NSCliving)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed test item treated tissues were used to avoid a possible double correction for colour interference.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. 

The test item Acid Red 143 showed significantly reduced cell viability in comparison to the negative control [true tissue viability (TTV%): 44 %]. All obtained test item viability results were below 60% when compared to the viability values obtained from the negative control.

Positive control viability results (mean tissue viability: 24 %) were below 50% when compared to the viability values obtained from the negative control. Furthermore, the mean OD value of the two negative control tissues (mean OD value: 1.342) were between 0.8 and 2.8. So the positive and negative controls showed the expected values within acceptable limits.The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item Acid Red 143 indicated that the test item has a toxic effect. The outcome of this study must be interpreted taken into account the outcome of the study carried out according to the OECD 438.

* TODTT: OD true MTT metabolic conversion for treated tissues

Justification for classification or non-classification

Skin irritation

The mean tissue viabilities obtained for the three experiments, conducted as per the OECD 439, is 124 % therefore above the threshold for skin irritation potential (50 %). The test substance is then considered non-skin irritant as per the CLP Regulation (EC) No. 1272/2008.

Eye irritation

Based on the tissue viability (44.0 %) determined in the study conducted according to the OECD Guideline 492 the substance is classified as a Cat.1 or Cat.2. Considering the results of the study conducted according to the OECD Guideline 438, no prediction can be made as the the overall ICE class was 1xII, 2xIII. From the results of the OECD 438 study, an eye damage potential of the substance can be excluded since. Taking into account both studies, the substance is classified as an Eye Irritant (H319), Cat. 2 as per the CLP Regulation (EC) No. 1272/2008..