Registration Dossier

Administrative data

Description of key information

not skin irritating

serious damage to eyes (Category 1)

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

The following data were obtained for Similar Substance 01. It is expected that the Target Substance will present similar skin and eye irritation potential to Similar Substance 01. Justification for the use of a Read Across approach is provided in Section 13 of IUCLID.

SKIN CORROSION/ IRRITATION

The potential of Similar Substance 01 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™, according to the the OECD Guideline 439 (2015). Similar Substance 01, 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 in the test system being this reaction an index of cell viability. A preliminary test was carried out to evaluate the compatibility of Similar Substance 01 with the test system: for the abilities of reducing MTT and of colouring water per se. In the Main Assay, Similar Substance 01 was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 mg/cm^2). Positive and negative controls (sodium dodecyl sulphate and D-PBS, respectively) were concurrently tested at the treatment level of 20 µL/epidermis unit. In order to verify whether Similar Substance 01 results needed 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 Similar Substance 01 is a dye and 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.

All the validity criteria were met. The colouring interference (NSC) was 1 %, while the non specific MTT reduction (NSMTT) was -1 %; thus only the blank subtraction was performed. Similar Substance 01 did not induce cell death in any replicate; the mean cell viability after the blank subtraction was 104 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.6.

Similar Substance 02 also demonstrated no potential for skin irritation in the EPISKIN™ test, as the average viability of tissues treated with Similar Substance 02 was found to be 94.0 % of the negative control average value (i.e. was greater than 50 %) in an experimental study according to the OECD Guideline 439 (201). Similar Substance 03 also demonstrated no potential for skin irritation, however was tested in vivo in an experimental study which examined the potential of a topical application of Similar Substance 03 to shaved dorsal rabbit skin to induce erythema and oedema, according to the OECD Guideline 404 (1987). The presence of no skin irritation potential in experimental studies across the board supports the use of the read-across approach for estimating the skin irritation potential of the Target Substance.

 

SERIOUS EYE DAMAGE/EYE IRRITATION

Two experimental studies were performed in order to evaluate the potential for Similar Substance 01 to induce eye irritation or serious eye damage. First, the EpiOcularTM Eye Irritation Test was performed in order to determine whether or not Similar Substance 01 was capable of inducing eye irritation or serious eye damage, without differentiation. Based on these findings, it was necessary to perform a more sensitive test, the Bovine Corneal Opacity Permeability (BCOP) Test, which was able to determine to what extend Similar Substance 01 caused eye damage or irriation, and which classification was necessary, either Category 1: Serious Eye Damage or Category 2: Eye Irritation. The results of this test demonstrated that Similar Substance 01 was indeed capable of causing serious eye damage.

EpiOcularTM Test: in order to evaluate the potential of Similar Substance 01 to provoke eye irritation in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study, the EpiOcularTM Eye Irritation Test was performed, according to the OECD Guideline 492 (2015). Similar Substance 01 was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to each tissue. After treatment, the 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. As Similar Substance 01 is a dye with an inherent dark colour, additional tests for data correction were performed to exclude colour interference during the photometric measurement.

After treatment with Similar Substance 01, the mean value of tissue viability was 3.0 %. All the validity criteria were met.

This value is below the threshold for eye irritation potential (≤ 60%). Under the conditions of the test, Similar Substance 01 was considered either eye irritating or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

In order to assess the corneal damage potential of the substance, an in vitro study was performed by quantitative measurements of changes in opacity and permeability in a bovine cornea according to the OECD Guideline 437 (2013) and EU Method B.47. The bovine corneas used were collected from slaughtered cattle which were between 12 and 60 months old. The test item was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Two valid experiments were performed. The result of the first experiment was declared equivocal, because one of the three replicates was an outlier. Therefore the experiment was repeated.

The negative control (HBSS solution) and the positive control (20% imidazole solution) met the validity criteria. Under the conditions of this study, the test item induced serious eye damage in the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 89.92 in the first experiment and 62.06 in the second experiment.

BCOP Test: in order to assess the corneal damage potential of Similar Substance 01, an in vitro study was performed by quantitative measurements of changes in opacity and permeability in a bovine cornea according to the OECD Guideline 437 (2013) and EU Method B.47. The bovine corneas used were collected from slaughtered cattle which were between 12 and 60 months old. Similar Substance 01 was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. Similar Substance 01 was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Two valid experiments were performed. The result of the first experiment was declared equivocal, because one of the three replicates was an outlier. Therefore the experiment was repeated.

The negative control (HBSS solution) and the positive control (20% imidazole solution) met the validity criteria. Under the conditions of this study, Similar Substance 01 induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 89.92 in the first experiment and 62.06 in the second experiment.

Similar Substance 02 also demonstrated the potential to cause eye irritation/eye damage in an EpiOcularTM Eye Irritation Test according to the OECD Guideline 492 (2015): Similar Substance 01 demonstrated 4.6 % optical denstiy at 570 nm of the negative control value, i.e. was less than 60 %, therefore it was considered to be capable of inducing either eye irritation or damage. A subsequent in vivo test was performed according to the EU Method B.5 (2017) whereby 0.1 g of Similar Substance 02 was applied to the left eye of three rabbits for a duration of one hour, after which the animals were monitored for 72 hours for corneal opacity, iritis, conjunctival redness and chemosis. Similar Substance 02 was not found to be irritating to the rabbit eye in vivo, and any minor symptoms observed were due to the mechanical irritation effect of the solid powder rather than its inherent capacity to cause damage or irritation. Similar Substance 03 was also tested in vivo in a topical application of 0.1 g to the left eye of three rabbits, according to an earlier version of the EU Method B.5 (1684) and according to the OECD Guideline 405 (1987). Similar Substance 03 demonstrated slightly higher corneal opacity and conjunctivae redness scores than Similar Substance 02, which determined that it was indeed an Eye Irritant. The presence of eye irritation/eye damage potential in all three similar substances across the board supports the use of the read-across approach for estimating the eye irritation potential of the Target Substance.

Justification for classification or non-classification

SKIN CORROSION/ IRRITATION

According to the CLP Regulation (EC 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 104 % when compared to the negative control. Thus, no classification for the skin irritation is warranted according to the CLP Regulation (EC 1272/2008).

SERIOUS EYE DAMAGE/EYE IRRITATION

According to the CLP Regulation (EC 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 substance, the mean value of tissue viability was 3.0 %. 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 89.92 in the first experiment and 62.06 in the second experiment. As both values are > 55 , the substance should be classified in Category 1 (irreversible effects on the eye) according to the CLP Regulation (EC 1272/2008).