Registration Dossier

Administrative data

Description of key information

not skin irritating

not eye irritating

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:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION/CORROSION

The skin irritation potential of the test item was evaluated in an in vivo experimental study equivalent to the OECD Guideline 404 (1987). 1 ml of test item diluted in WEM was applied to the clipped skin of three male, New Zealand White rabbits for 4 hours using an occlusive dressing, and skin was monitored for erythema and oedema at 30 minutes, 24, 48 and 72 hours.

The mean (24/48/72 hours) erythema scores were 0 in all animals; mean (24/48/72 hours) oedema scores were 0 in animal #1, and 0.33 in animals #2 and #3, and oedema was reversible within 48 hours.

The skin irritation results of Acid Black 076 can be supported by experimental data on Similar Substance 01 and Similar Substance 02.

The potential for Similar Substance 01 to cause dermal corrosion/irritation was assessed in an in vitro experimental study according to the OECD Guideline 431 (2013) (a Skin Corrosion Test (SCT)), and the OECD Guideline 439 (2013) (a Skin Irritation Test (SIT)). A single topical application of 25 µl (about 12 mg) of undiluted test item was applied to a reconstructed, three-dimensional human epidermis model (EpiDerm™). For the corrosion test, two EpiDerm™ tissue samples were incubated with the test item for 3 minutes and 1 hour, respectively. For the irritation test, three EpiDerm™ tissue samples were incubated with the test item for 1 hour followed by a 42-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

Due to the intense color of the test item, it could not be determined whether the test item was able to reduce MTT directly. Therefore, an additional MTT reduction control was introduced. However, the result of the control tissues inactivated by freezing did not indicate an increased MTT reduction. In both tests, minimal compound residues remained on the tissues after the washing procedure, however, this did not interfere with the colorimetric test as was demonstrated in a pretest. In the corrosion test, the mean viability of the test item-treated tissues determined after an exposure period of 3 minutes was 117 %, and it was 101 % after an exposure period of 1 hour. In the irritation test, the mean viability of the test item-treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 105 %. Based on these observations, it can be concluded that the test item does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the reported test conditions.

The skin irritation potential of Similar Substance 02 was evaluated in an in vivo experimental study equivalent to the OECD Guideline 404 (1987). The entire back and flanks of 3 male and 3 female Himalayan rabbits were shaved with an electric clipper, and immediately before treatment one side was slightly scarified (abraded) and the other side remained intact. A gauze patch (2.5× 2.5 cm) laden with test item was applied to each side of each rabbits back for 24 hours with an impermeable over, and fastened around the body for a duration of 24 hours. The skin was monitored for erythema and oedema at 24, 48 and 72 hours, 4 and 7 days after removal of the gauze patches.

The mean (24/48/72 hours) erythema and oedema scores were both 0.67 in all animals for the intact application. Erythema and oedema were both reversible with 72 hours.

EYE IRRITATION

The eye irritation potential of the test item was evaluated in an in vivo experimental study equivalent to the OECD guideline 405 (1987). Three male rabbits were administered 100 mg of undiluted test item to the conjunctival sac of the right eye and not removed; the left eye served as a control. The eyes were evaluated for corneal opacity, iritis, conjunctival redness and chemosis at 30 minutes, 24, 48 and 72 hours after administration.

The corneal opacity and iritis scores were 0 in all animals at all time points. The mean (24/48/72 h) chemosis scores were 0 in animals #1 and #3, and 0.33 in animal #2. Mean (24/48/72 h) conjunctival redness scores were 0, 0.33 and 0.67 in animals #1, #2 and #3 respectively. All effects observed were fully reversible within the 7-day study period.

The eye irritation results of Acid Black 076 can be supported by experimental data on Similar Substance 01 and Similar Substance 02.

Similar substance 01 was tested in the EpiOcular™ in order to define its eye irritation potential. 50 μl bulk volume (about 15 mg) of the undiluted test item was applied to a reconstructed 3D human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test item for 6 hours followed by an 18-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as the endpoint. The formazan production of test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

Due to the intense inherent color of the test item, it could not be determined whether the test item was able to reduce MTT directly. Therefore, an additional MTT reduction control was introduced. The result of the control tissues inactivated by freezing (killed control, KC) indicated an increased MTT reduction. Compound residues remained on the tissues after the washing procedure, however, this did not interfere with the colorimetric test as was demonstrated in a pretest. The final mean viability of the test item-treated tissues was 78 %. Therefore, the substance is not classified as eye irritant.

The eye irritation potential of Similar substance 02 was evaluated in an in vivo experimental study equivalent to the OECD Guideline 405 (1987). 0.1 g of test item was applied to the left conjunctival sac of 3 male and 3 female Himalayan rabbits and the lids wer gently held closed for one second. The right eye served as an untreated control. The treated eye of three of the six rabbits (female rabbits only) was flushed with 10 ml of lukewarm water approximately 30 seconds after application. Treated eyes were monitored for cornea (opacity and area), iris and conjunctivae (redness, chemosis and discharge) using a slit-lamp on days 1, 2, 3, 4 and 7 after application.

Cornea, iris and conjunctivae scores were 0 for all animals at all time points monitored. Therefore, the substance is not eye irritant.

Justification for classification or non-classification

SKIN IRRITATION/CORROSION

According to the CLP Regulation (EC) no. 1272/2008, substances that 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:

- 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, the mean (24/48/72h) erythema and oedema scores were not in 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).

 

EYE IRRITATION

According to the CLP Regulation (EC no. 1272/2008), substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) if, when applied to the eye of an animal, a substance produces a positive response in at least 2 out of 3 animals:

- corneal opacity ≥ 1, and/or

- iritis ≥ 1, and/or

- conjunctival redness ≥ 2, and/or

- conjunctival oedema (chemosis) ≥ 2

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test item, and which fully reverses within an observation period of 21 days.

 

Based on the results obtained in in vivo the eye irritation study performed, no classification of the test item is warranted for Serious eye damage/eye irritation according to the CLP Regulation (EC) no. 1272/2008.