Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Endpoint summary

Description of key information

Non skin irritant.

Eye corrosive.

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 (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The corrosion potential of the substance to the skin was assessed in-vitro according to the OECD Guideline 431. The substance was applied topically to a three-dimensional human skin model, comprising a reconstituted epidermis with a functional stratum cornneum. Corrosivity was identified by the ability of the substance to produce a decrease in cell viability (by quantification of formazan production). For this reason, the tissues, after the removal of the substance, were incubated with MTT medium and the optical density of the extracted formazan was measured at 570 nm. Negative and positive controls run in parallel; the % photometric absorption of test item and positive control was calculated by comparison with the negative control. Relative absorbance were increased to 103 % after three minutes and were decreased to 74.3 % after one hour treatment with the substance. These values are laying above the threshold for corrosivity i.e. < 50 % of negative control after 3 min or > 50 % of negative control after 3 min and < 15 % of negative control after 1 hour. The substance is not considered to be a skin corrosive.

The skin irritation potential of the test item was evaluated by using the Episkin reconstructed human epidermis model, according to the OECD Guideline 439, EU Method B.46 and standard operating procedure version 1.2: EpiskinTM Skin irritation test 42 hours Determination of IL-1α concentration in the culture medium, published by ECVAM. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at 37 °C, 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. In addition, the concentration of the inflammatory mediator IL-1α was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50 % following the MTT reduction assay. All acceptance criteria for the negative and positive controls were fulfilled. The relative mean viability of the tissues treated with the test item was 99 % with a standard deviation of 3 %. As the mean relative viability > 50 % after the MTT reduction, the IL-1α concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1α concentration value of one tissue was found below the LOQ (< 5.00 pg/ml). Consequently, the mean IL-1α concentration from the three test item-treated tissues was not calculated. The IL-1α concentration values of the two other test item-treated tissues were found < 60 pg/ml (6.17 and 9.89 pg/ml). Therefore, the substance is not considered to be a skin irritant.

Eye irritation

The irritation potential of the substance to the eyes was assessed in the in-vitro study by detection of damages in blood vessels under the chorionalantoic membrane (CAM) of incubated chicken eggs (nine days old). The reactions on the CAM were observed over a period of 300 seconds and the time for the appearance of haemorrhage, lysis and coagulation were recorded in order to estimate the irritation score. Negative control and two positive controls run in parallel. Both positive controls induced severe irritation on the blood vessels whereas the negative control showed no irritation. Both positive and negative control data lay within the accepted historical data. A mean IS of 17.69 was calcualated for the test substance. The substance is considered to be as corrosive to the eye.

The potential irritant and corrosive properties of the test item to the eye was further evaluated in the BCOP assay, according to the OECD Guideline 437. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item was applied 20 % (w/v) in the vehicle (0.9 % NaCl), in a single experiment using a treatment time of 4 hours and using the closed-chamber treatment method. Vehicle and positive controls were concurrently testes. 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 at 32 °C. At the end of the incubation period, the OD 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.

Opacity and fluorescein fixation were observed on the corneas treated with the test item. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 47.

As the mean IVIS was > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (No Category).

Justification for classification or non-classification

The substance was assessed for its skin and eye irritation potential in two in-vitro studies.

Skin irritation

In the OECD 431 assay, the substance did not induce any decrease in cell viability (quantified by formazan production). At three minutes the cell viability was 103 % and at one hour was 74.3 %. These values lay above the thresholds of corrosivity, i.e. < 50 % of negative control after 3 min or > 50 % of negative control after 3 min and < 15 % of negative control after 1 hour. The substance is not considered as a skin corrosive.

In the OECD 439 assay, the mean viability of the tissues after the exposure to the substance was 99 % and and mean IL-1α released concentration ≤ 60 pg/ml. The viability was greater than 50 % and thus the substance is not considered as a skin irritant.

According to CLP Regulation guidance, the classification criteria for the in-vitro skin corrosion/irritation studies are mentioned in the corresponding OECD test guideline. Based on the results of the in-vitro studies and the reccomendations of the OECD Guideline 431 and 439, the substance should not be classified for skin corrosion nor skin irritation. The substance is not classified for skin irritation according to the CLP Regulation (EC) No. 1272/2008.

Eye irritation

In the BCOP test the mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 47. As the mean IVIS was > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (No Category).

During the HET-CAM test, the substance has an irritation score of 17.69 based on the time of the appearance of haemmorhage, lysis and coagulation signs. Irritation scores greater than nine permit the severe irritancy classification. The results of this study do not permit the distinction between irritant and corrosive substances.

According to the CLP Regulation, positive results in the HET-CAM study can be used for classification purposes i.e. leading to a classification of Category 1 for serious eye damage. In addition, the IVIS score determined in the BCOP study is between 3 -55 which means that no prediction can be made. However, based on the results of the two in-vitro studies the substance should be classified in Category 1 for serious eye damage according to the CLP Regulation (EC) No.1272/2008.