[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin Irritation/Corrosion

Skin irritant

Eye Irritation/Corrosion

Eye corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation/Corrosion

In Vitro Skin Corrosivity Test in the EPISKINTM(SM) Model

An in vitro skin corrosivity test was performed with the test item in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline (2016).

In each experiment, disks of EPISKINTM(SM) (at least two units) were treated with powdered test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

In this study two experiments were performed for the scientifically valid data. Two test item treated skin units, two NSCliving and NSCkilled control tissues and two additional controls on killed epidermis (two test item treated and two negative control treated skin units) were used in Experiment I. In Experiment II five test item treated skin units and additional controls on killed epidermis were used, but no colour control disks were used (as they were not necessary based on the results of the first experiment).

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

In the Experiment I the individual viability values of the two replicates were 42.3% (indicating a non-corrosive result) and 31.7% (indicating a corrosive result) compared to the negative control value. Therefore, an additional experiment (Experiment II) was performed using the same experimental conditions (but using increased number of replicates) to clarify the result and to provide proper information for prediction. In the Experiment II the mean cell viability of the test item treated skin samples showed a negative result.

Following exposure with the test item, the mean cell viability was 37.0% in Experiment I and 57.9% in Experiment II compared to the negative control (after adjustment for non-specific MTT reduction). Mean value of all viability data (51.9%) was above the threshold limit of 35%, therefore the test item was considered as being non-corrosive. The experiments met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with the test item, the results indicate that the test item is non-corrosive to the skin.

In vitro skin irritation on EPISKIN(TM) Reconstructed Human Epidermis

The objective of this study was to evaluate the skin irritation potential of the test item using the Episkin^TMreconstructed human epidermis model. The study design was based upon following guidelines:OECD Test Guideline No. 439.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes (±1 minute). At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +, 5% CO₂in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (as reference viability).

In the preliminary tests, the test item was either found to have direct MTT reducing properties, and colouring potential.

All acceptance criteria were fulfilled in both validated main tests (with one exception for the SD value of the % viability between test item-treated tissue replicates in the second main test), the study was therefore considered to be valid. 

Following the 15-minute exposure and the 42-hour recovery period, the true relative mean viabilities of the tissues treated with the test item were 48% and 43% with a Standard Deviations of 10% and 20%, for the first and the second main tests, respectively. Both validated main tests presented borderline results, since the true relative mean viability values were close and below the range of 50%, with non-concordant individual viability values (i.e.distributed on both sides of the 50% threshold).

These borderline results were therefore considered related to the test item itself, thus no third run was perfomred.

Based on the overall viability results of the study and since both mean viabilities were < 50% after the MTT reduction,the results met the criteria for an irritant response.

Under the experimental conditions of this study, the test item is considered to be irritant to skin.

Eye Irritation/Corrosion

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, to the eye. The design of this study was based on the OECD Test Guideline 437.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and vehicle control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, applied at the concentration of 20% (w/v) in the vehicle (i.e.NaCl 0.9%) and both vehicle and positive controls were tested using a treatment 4 hours(± 5 minutes)and the closed‑chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. A second opacity measurement was then performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at + 32 °C.

At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Macroscopic examination

Opacity, fluorescein fixation and blue colouration of the corneas were observed all corneas treated with the test item formulation.

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the corneas treated with the test item formulation was 118.

Under the experimental conditions of this study, the test item was identified asinducing serious eye damage.

Justification for classification or non-classification

Skin Irritation

Accepted in vitro test methods to detect skin corrosion/irritation (i.e. Category 1 and 2 under CLP) and/or absence of effects (i.e. not classified under CLP) have been applied to assess the skin irritation/corrosion potential of the test substance, according to the Integrated Approach on Testing Strategy (IATA).

Considering that the substance is a vanadyl derivative, and therefore with an intrinsic high potential to induce toxic effect to the skin, the first selection was the OECD guideline 431.

Under test condition the test substance resulted non-corrosive to the skin. However this test method alone, as reported in ECHA guidance R.7a (v.6.0, July 2017), cannot detect wheter the substance is skin irritant or not skin irritant.

For this reason a second experiment was carried out according to the OECD guideline 439.

In reference to the CLP Regulation (EC 1272/2008), results of this test method may be considered as follows:

- non irritant to skin, if tissue viability after exposure and post-treatment incubation period is > 50 %,

- category 2 or category 1, if the mean percent tissue viability after exposure and post-treatmen incubation is ≤ to 50 %.

Under test condition the substance shows a mean viability between 43 % and 48 % therefore, considering that the substance is non corrosive according the OECD guideline 431, it has been classified as Skin Irritant Cat.2 according to the CLP Regulation n. 1272/2008.

Eye Irritation

The ECHA Guidance on the application of the CLP Criteria set out that a substance can be considered as causing serious eye damage (Category 1) based on positive results in the BCOP test, while negative results from studies carried according to the BCOP test method can be used for classification purposes, in an integrated approach strategy (IATA) .

According to the OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), the IVIS cut-off values are the follows:

 

- IVIS ≤ 3 – No Classification (UN GHS)

- IVIS > 3; ≤ 55 – No prediction can be made on the non classification

- IVIS > 55 – Category 1 (UN GHS)

 

The test item showed a value of in vitro irritancy score (IVIS ) of 118, therefore the test substance has been classified as Corrosive to Eye Category 1, according to the CLP Regulation n. 1272/2008.