Reaction mass of Cobalt, 4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1Hpyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate 4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-1-yl]benzenesulfonate sodium complexes and Cobaltate(3-), bis[4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-1-yl]benzenesulfonato(3-)]-, trisodium and Cobaltate(5-), bis[4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1Hpyrazol-1-yl]benzenesulfonato(4-)]-, pentasodium

Endpoint summary

Description of key information

Not a skin irritant/corrosive

Category 1: Serious Eye Damage

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 AND CORROSION

The skin irritation potential of the substance was evaluated by considering data on the substance itself as well as data on Source 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 test item was evaluated in an in vivo experimental study. The test item was applied to both the intact and abraded skin of six rabbits (three males and three females). The mean (24/48/72 hour) erythema and oedema scores were found to be 2.3 or lower in all animals. Moreover, reversibility of all effects was observed within 72 hours after application to the intact skin; reversibility of all effects was observed in four of the six animals within 72 hours after application to abraded skin: erythema (grade 1) to abraded skin was present in animal #3 at 72 hours, and oedema (grade 1) to abraded skin was present in animal #1 at 72 hours.

The skin irritation potential of a structural analogue, Source Substance 01, 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, with the exception of one animal (#2) which showed signs of erythema and oedema (grade 1 for both erythema and oedema at 24 hours and grade 2 erythema and oedema at 72 hours), 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. As the observations time were only made at 24 and 72 h, for mean calculations of 24/48/72 h, a worst-case approach was used: the higher score of either 24 h or 72 h was used to complete the 48 h score. The Source Substance was considered to be not irritating to both intact and abraded rabbit skin.

Both the test item and Source Substance 01 demonstrated an absence of skin irritating potential when applied topically to intact and abraded rabbit skin. As these results were cohesive for in vivo skin irritation, the read-across analogue approach can be considered an appropriate and the test item can be considered not irritating.

EYE IRRITATION AND CORROSION

Three experimental studies were available for the assessment of the eye irritation/damage potential of the test item: an EpiOcular^TM test, a Bovine Corneal Opacity and Permeability (BCOP) test and an in vivo study conducted on the rabbit eye with insufficient documentation for evaluation (R4).

EpiOcular^TM Study

In order to evaluate the potential of the test item to evoke eye irritation in a Reconstructed human Cornea-like Epithelium (RhCE) model in anin vitrostudy, 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.

Bovine Corneal Opacity and Permeability Study

The potential for the test item to induce eye irritation and damage was evaluated 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. Mean in vitro irritation scores (IVIS) 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 irriversible serious eye damage.

In Vivo Eye Irritation / Corrosion Study

Eye irritation potential of the test item was evaluated by a topical application to the left eye of six rabbits (three males and three females). Under the conditions of this study, the test item resulted in adverse effects on the conjunctivae: the mean values at 24/48/72 hours were ≥ 2 for redness in five animals (all except animal #5), while the mean values for chemosis were ≤ 2 in all animals. The mean values at 24/48/72 hour intervals for corneal opacity were ≤ 1 in all animals, except for one animal (animal #1). The iritis scores were 0 in all animals.

Reversibility of corneal opacity was observed 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 0 at the end of the study period in two animals (#1 and #6) and in all animals respectively, however the study was terminated at 72 hours instead of the recommended 21 days. Nevertheless it is worth saying that an improvement trend generally was noted, as a decreasing in the conjunctivae scores was observed during the 72 hour study period.It should be noted that documentation of this study was considered insufficient for evaluation. The test item was considered slightly irritating to the rabbit eye in vivo.

All three tests demonstrated positive results for either eye irritation or damage. The result of the BCOP test demonstrates that the test item causes irreversible Serious 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 studies conducted on the test item and Source 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 on the substance, the calculated mean IVIS (in vitro irritancy score) was 106.94 . As the value is > 55 , the test item is classified in Category 1: Serious Eye Damage (irreversible effects on the eye) according to the CLP Regulation (EC) No. 1272/2008.

Finally, it is worth noting that the in vivo test results available can confirm the eye irritatio/corrosion potential (slight irritant to rabbit eye).