Cobaltate(3-), bis[4-[4-[[4-[4-[[5-(aminosulfonyl)-2-hydroxyphenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(3-)]-, trisodium

Endpoint summary

Description of key information

Not irritating

Category 1: Serious Eye Damage

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Irritation / Corrosion

The skin irritation potential of the substance was evaluated by considering data on the substance itself as well as data on Similar Substance 01 following a weight of evidence approach. This decision was made considering the low purity of both test items used experimentally, the high similarity between the substances and the coherence in results. Justification for the use of a Read Across approach is provided in Section 13 of IUCLID.

The skin irritation potential of the substance was evaluated in an in vivo skin irritation test performed using the intact and abraded skin of six rabbits, according to the ETAD Method No. 002. Erythema and oedema scores were found to be 0 in five of the animals, both on the intact and abraded skin sites. Only one animal (#2) shown signs of erythema and oedema (grade 1 for both erythema and oedema at 24 hours and grade 2 for erythema and oedema at 72 hours), and these signs were only present on the intact skin. These data should be considered ambiguous due to the fact that no erythema or oedema was observed on the abraded skin site of the same animal at the same time intervals. Moreover, the uncertainty of these data can be confirmed considering that no effects were observed in all the other animals.

The potential of to induce skin irritation or corrosion was evaluated in an experimental study on three male and three female rabbits. The test was performed on both intact and abraded skin. The mean values of erythema and oedema scores at 24/48/72 h were greater than or equal to 2.3 in all animals. Reversibility of all effects within 72 hours was observed on the intact skin, whereas reversibility of effects was observed also on abraded skin, with the exception of erythema (grade 1) on animal #3 and oedema (grade 1) on animal #1.

Based on this weight of evidence approach, the substance cannnot be considered as skin irritating.

Eye Irritation / Corrosion

The eye irritation potential of the substance was evaluated by considering data on the substance itself as well as data on Similar Substance 01 following a weight of evidence approach.

The eye irritation potential of the substance was evaluated in an in vivo experimental study according to the ETAD Method No. 003, similar to OECD Guideline 405. 90 mg of test item was applied to one eye of 6 rabbits and was monitored for corneal opacity/area, iritis and conjunctival redness/chemosis/discharge for 7 days.

Under the conditions of this study, application of the test item resulted in no adverse effects on corneal opacity and iris: mean values at 24, 48 and 72 h for were 0 in all animals. Adverse effects on the conjunctivae resulted in 2 animals: mean values at 24, 48 and 72 h were 1.3 for redness in one animal and 1 for redness and 0.33 for chemosis in the other animal. These effects were reversible within 7 d in the first animal and within 72 and 48 h for redness and chemosis respectively in the other animal. The mean values for conjunctive in the other animals were 0.

Data on eye irritation potential of the test item were also obtained using a experimental studies on Similar Substance 01. Justification for the use of a Read Across approach is provided in Section 13 of IUCLID.

In order to evaluate the eye irritation/corrosion potential of the Similar Substance 01, two in vitro studies were performed (EpiOcular^TMEye Irritation and BCOP tests).

The EpiOcular^TMEye Irritation Test was performed according to the OECD Guideline 492 (2017). The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment, the test item was rinsed from the tissue and cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. 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 1.7. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 40.0 % (< 50 %).Variation within tissue replicates was acceptable (< 20 %).In the main test after treatment with the test item, the mean value of relative tissue viability was 2.9 %. In a pre-test, the test item showed intense coloring, there was a risk that the photometric measurement may be influenced. Normally, an additional test for intensely coloured test items should have been performed. This additional test was not performed as the result was already clearly positive (2.9 % viability) and only a false negative result is possible with coloured substances which absorb on the same wavelength as formazan, a false negative result is not possible anymore. The value of tissue viability obtained is well below the threshold for eye irritation potential (≤ 60%). Substances that induce values below the threshold are either an eye irritant or induce serious eye damage. Under the conditions of the test, the test item is considered either an eye irritant or inducing serious eye damage in the main test of the EpiOcular^TMEye Irritation Test.

The BCOP test was performed in an experimental study according to the OECD Guideline 437 (2017) and the EU Method B.47 (2017). Fresh bovine corneas were removed surgically from freshly slaughtered cattle (Bos primigenius Taurus) aged 12 to 60 months, and stored in cooled Hanks’ Balanced Salt Solution (HBSS) with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL)within 1 hour. The baseline opacity of each cornea was examined. Test item solution (1:10 test item in 20 % HBSS) diluted in demin. water) was applied to the epithelial side of three replicate corneas for a duration of four hours at 32 °C ± 1 °C. A negative control (HBSS alone; three replicates) and a positive control (20 % Imidazole solution in HBSS; three replicates) were tested in parallel. The anterior chamber of each cornea was subsequently washed with cMEM containing phenol red at least three times, then once with cMEM alone, and final corneal opacity values of each cornea in cMEM were examined. 1 ml of fluoricine was applied to the front chamber of each sample and incubated for 90 minutes at 32 °C, then the optical density of the contents of the posterior chamber at 492 mm was evaluated in order to calculate the corneal permeability. Meanin vivoirritation scores were derived for the test item, the negative control and the positive control. A substance which induces serious eye damage has an IVIS score higher than 55; whereas an IVIS value of > 3 and ≤ 55 indicates that no prediction of eye irritation and damage potential can be made.

The mean IVIS value of the test item was found to be 106.94 with a relative standard deviation of 35.64 %. The negative control was found to have a mean IVIS value of 1.74 with a relative standard deviation of 116.45 %. The positive control was found to have a mean IVIS score of 103.08 with a relative standard deviation of 7.60 %. Based on the IVIS scores, the test item was found to induce serious eye damage.

In addition, an in vivo experimental study performed on the Similar Substance 01 is available. The test item was applied on the eye of six rabbits (three males and three females) and the method followed was according to the ETA Method 003.

The mean (24/48/72 h) values were ≥ 2 for redness in five out of six animals (all except animal #5). The mean (24/48/72 h) values for chemosis were ≤ 2 in all animals. The mean (24/48/72 h) values for corneal opacity were ≤ 1 in five out of six animals (all except animal #1). The mean (24/48/72 h) iritis scores were 0 in all animals.The effects on corneal opacity were reversible in all animals, except for one (animal #1). The effects on conjunctivae were not fully reversible within 72 h observation period in all animals: chemosis and redness scores were not reversible by the end of the study period in two animals (chemosis) and in all animals (redness). Nevertheless it is worth noting that a trend of general improvement was noted: decreased conjunctivae scores were observed during the 72 hour study period.

In the available in vivo studies performed on both substances comparable observations were made and in both studies a test item with a low purity was used. The available data obtained in the in vitro studies performed using the Similar Substance 01 are more recent, obtained in GLP compliance and by using a sample with a higher purity. Considering the comparable observations of the in vivo tests, the high structural similarity between the substances, it could be expected that comparable results will be obtained in (the same) in vitro test by using the Target Substance. Therefore, by following also a conservative approach, the substance should be considered as to have the same potential to cause an eye damage.

Justification for classification or non-classification

SKIN IRRITATION/CORROSION

According to the CLP Regulation (EC) no. 1272/2008, substances which have the potential to induce reversible damage to the skin following the application of a substance for up to 4 hours are classified as Category 2 (irritating to the skin) if they present:

- a mean (24, 48 and 72 hour) erythema or oedema score of 2.3 to 4.0 inlcusive in at least 2 out of 3 tested animals or, if reactions are delayed, from scores on 3 consecutive days after the onset of skin reactions; or

- inflammation that persists to the end of the observation period (normally 14 days) in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling; or

- in some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

 

Based on the results of the in vivo skin irritation study conducted on the test item and Similar Substance 01, the mean (24/48/72 hour) erythema and oedema scores were not within the range of ≥ 2.3 to ≤ 4.0; therefore the test item is not classified for skin irritation 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 pre-existing 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 test item, the mean value of tissue viability was 2.9 %. 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 chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: IVIS UN GHS score ≤ 3 depicts no classification; values > 3 and ≤ 55 depict that no prediction can be made; values above 55 classify a substance as Category 1: Serious Eye Damage.

Based on the results obtained in the BCOP test performed, the calculated mean IVIS (in vitro irritancy score) was 106.94 . As the value is > 55 , the substance is classified in Category 1: Serious Eye Damage (irreversible effects on the eye) according to the CLP Regulation (EC) No. 1272/2008.