Reaction products of diazotized Dodecyl Aniline coupled with 8-acetylamine-1-hydroxynaphthalen-3,6-disulphonic Acid, sodium salts

Endpoint summary

Description of key information

not skin irritant

eye irritant (Category 2)

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 CORROSION/ IRRITATION

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures were based on the OECD Guideline for testing of chemicals no. 439. The test item, 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. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. A preliminary test was carried out to evaluate the compatibility of the test item with the test system. The value obtained for the Optical Density (OD) indicated that the test item has a potential interfering ability. Based on these results, additional controls were added in the Main Assay. In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 ± 2 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 20 µl/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSC) 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 item is 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 (NSC killed) was performed.

The colouring interference (NSC) was 1 %, as well as the non specific MTT reduction (NSMTT); thus only the black subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 105%, when compared to the negative control. All the validity criteria were met. Based on the results obtained, the test item is not classified as irritant to the skin (UN GHS No Category).

SERIOUS EYE DAMAGE/EYE IRRITATION

Firstly, an in vitro study was performed according to the OECD Guideline 492 (2015) in order to evaluate the potential of the test item to evoke eye irritation in a Reconstructed human Cornea-like Epithelium (RhCE) model.

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. As the test item is intensely coloured, an additional test for colour interference was performed to exclude wrong photometrical measurement values due to intense colour of the test item. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.6. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 42.2 % (< 50 %). Variation within tissue replicates was acceptable (< 20 %). After treatment with the test item, the mean value of relative tissue viability was 36.6 %. This value is below the threshold for eye irritation potential (≤ 60 %). The EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).

Based on this result, the test item is considered either eye irritant or inducing serious eye damage and further investigations were needed.

Therefore, a second in vitro study was performed according to the OECD Guideline 437 (2017) andEU Method B.47 (2017). The test item was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured. The test item was tested pure.

Under the conditions of this test, the test item showed effects on the cornea of the bovine eye. The calculated IVIS is 12.14.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category (“no predicition can be made”).

The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Justification for classification or non-classification

SKIN CORROSION/ IRRITATION

According to the CLP Regulation (EC) no. 1272/2008, skin irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours. In vitro alternatives that have been validated and accepted may also be used to help make classification decisions. Table R.7.2-2 in the Guidance on IR&CSA lists the status of validation and regulatory acceptance for in vitro test methods for skin corrosion and skin irritation. The OECD has adopted an in vitro skin irritation test guideline i.e. OECD TG 439 (TM B. 46) that currently contains four test methods including the EpiSkinTM. This test method allows distinction between irritants (CLP Cat 1/Cat 2) and substances not classified. Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

Based on the results obtained in the test performed on the substance, the mean cell viability after the blank subtraction was 105 % when compared to the negative control. Therefore, no classification for the skin irritation is warranted according to the CLP Regulation (EC) no. 1272/2008.

SERIOUS EYE DAMAGE/EYE IRRITATION

According to the CLP Regulation (EC) no. 1272/2008, serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application. The classification system for substances involves a tiered testing and evaluation scheme, combining preexisting information on serious ocular tissue damage and on eye irritation as well as considerations on (Q)SAR and the output of validated in vitro tests in order to avoid unnecessary animal testing.

The OECD has at present adopted five in vitro test guidelines for assessing eye hazard potential. These tests are recommended for use as part of a tiered-testing strategy for regulatory classification and labelling.

Test method OECD TG 492 – Reconstructed human Cornea-like Epithelium Test Method (RhCE) is an in vitro assay that may be used to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage. The only in vitro test method currently covered by this Test Guideline is the EpiOcular™ Eye Irritation Test (EIT). The percentage tissue viability cut-off value for identifying test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) is > 60 %.

Based on the results obtained in the RhCE test performed on the substance, the mean value of tissue viability was 36.6 %. This value is below the threshold for eye irritation potential (≤ 60 %). Thus, no prediction can be made and further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

Test method EU B.47 / OECD TG 437 – The Bovine Corneal Opacity and Permeability Test Method (BCOP) is an in vitro assay that may be used to identify chemicals (substances or mixtures) as either i) causing “serious eye damage” (Cat 1 of CLP), or ii) not requiring classification for eye irritation or serious eye damage according to the CLP. The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: IVIS UN GHS ≤ 3 No Category; > 3 and ≤ 55 No prediction can be made; > 55 Category 1.

Based on the results obtained in the BCOP test performed on the substance, the calculated IVIS (in vitro

irritancy score) was 19.14, 4.70 and 12.57 in the three experiments. The mean IVIS is 12.14 and the experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item. The test item induced an IVIS between 3 and 55, therefore „no predicition can be made with the BCOP“.

However, with this result Category 1 for serious eye damage can be excluded and in combination with the result of the EpiOcular study the test item is classified in Category 2 for eye irritation, according to the CLP Regulation (EC) no. 1272/2008.