Disodium naphthalene-1,5-disulphonate

Endpoint summary

Description of key information

The available information does not point to a skin sensitization potential in vivo. No protein binding alert was obtained via in silico assessment with the OECD Toolbox. The substance was tested in chemico for the first key event of skin sensitization (peptide reactivity) and in vitro for the second key event (keratinocyte activation). Both tests revealed negative results. A third test (activation of dendritic cells) is ongoing.

The above listed tests and procedures are adequate for non-classification regarding skin sensitization and risk assessment according to point 8.3 of Annex VII of Regulation (EC) 1907/2006.

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Information on available animal data for the assessment of skin sensitization:

No animal studies are available for the substance to assess the endpoint skin sensitization.

Information on human data for the assessment of skin sensitization:

No human data are available for the substance to assess the endpoint skin sensitization. However, no skin irritating or skin sensitizing effects were reported in occupational settings.

Information on physico-chemical properties:

Disodium-naphthalene-1,5-disulphonate is a solid with a molecular weight of 332 g/mole. The substance well soluble in water and a log Kow (octanol/water) of -3.5 was estimated.

Molecular weight: 332 g/mole

Appearance: solid

Log Kow (octanol/water): -3.5 (EPI-Suite v4.11, 2018)

Water solubility: 108 g/L (Currenta 2016/0021/04)

Information on dermal bioavailability:

No information is available on dermal bioavailability of the substance. Based on the physical-chemical parameters given above dermal absorption can be expected to be low (ECHA Guidance on Information Requirements, Chapter R7c, 2017).

In silico information - Structure Activity Relationship (QSAR via OECD Toolbox):

As in silico tool for the prediction of skin sensitization the QSAR OECD Toolbox 4.0 was used.

The outcomes of the mechanistic/endpoint relevant profilers for skin sensitization potential are:

Protein binding alerts:

No protein binding alerts were identified by OASIS v1.4 and OECD for the substance.

Protein binding potency:

Not possible to classify (Remark: It could be noted that the protein-binding potency profiler only predicts the reactivity quantitatively if the protein-binding mechanism is of Michael addition-type or Nucleophilic substitution type 2.).

Specific profilers for protein binding:

No activity in the Direct Peptide Reactivity Assay (DPRA) with regard to cysteine and lysine peptide depletion and no h-CLAT activity were foreseen. With regard to keratinocyte gene expression the substance is out of mechanistic domain.

In chemico testing - Direct Peptide Reactivity Assay (DPRA following OECD TG 442C):

Disodium-naphthalene-1,5-disulphonate was tested as monohydrate in the Direct Peptide Reactivity Assay (DPRA) following OECD 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA); adopted February 2015). This test addresses the first key event of skin sensitization, the covalent binding to skin proteins (haptenation). Protein binding is measured in chemico by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a cysteine/lysine prediction model to categorize a substance in one of four classes of reactivity:

• ‘no or minimal reactivity’ (DPRA prediction negative): ≤ 6.38% depletion

• ‘low reactivity’ (DPRA prediction positive): > 6.38 to ≤ 22.62% mean depletion

• ‘moderate reactivity’ (DPRA prediction positive): > 22.62 to ≤ 42.47% mean depletion and

• ‘high reactivity’ (DPRA prediction positive): > 42.47 to ≤ 100% mean depletion

The test item showed no relevant depletion of peptides in the DPRA with a cysteine/lysine depletion rate of 1.08% (Schaub M, 2018). The obtained value counts for a DPRA prediction of ‘minimal reactivity – negative’. This result is in line with the prediction of the QSAR Toolbox profiler for DPRA Cys or Lys binding potency (“not reactive”; within applicability domain).

In vitro testing in a first cell-based test – KeratinoSens test (following OECD TG 442D):

Disodium-naphthalene-1,5-disulphonate was tested as monohydrate in the KeratinoSens assay, an in vitro skin sensitization test (ARE-Nrf2 Luciferase Test Method) following OECD TG 442D (In vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method; adopted February 2015). This test addresses the second key event of skin sensitization, the activation of keratinocytes in cell culture. Skin sensitizers are reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic items such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes.

The ARE-Nrf2 luciferase test method utilizes an immortalized adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

A prediction in the KeratinoSens is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions (Imax >1,5-fold; cell viability > 70%, EC1.5 value < 1000 µM, apparent dose response curve).

All assay acceptance criteria were met. Since the Imax in both experiments was less than 1.5-fold, the test item was considered to be negative in the ARE-Nrf2 Luciferase Test.

In vitro testing in a second cell-based test - h-CLAT (following OECD TG 442E)

The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization test following OECD TG 442E (In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT), adopted July 2016). This test addresses the third key event of skin sensitization, the activation of dendritic cells in culture. In principle, changes in the expression of cell surface markers (i.e. CD86 and CD54) on a human monocytic leukaemia cell line are quantified following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic dendritic cell (DC) activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control is calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

Testing of disodium-naphthalene-1,5-disulphonate monohydrate in the h-CLAT is ongoing.

Further toxicological data with potential relevance for skin sensitization:

The available toxicological data are of reliability 1 or 2 (Guideline- and GLP- compliant studies; see Table 3). No effects with regard to skin or eye irritation were observed in vitro and in vivo.

The absence of an electrophilic (DNA-reactive) potential of the substance was shown in a bacterial mutagenicity test with and without metabolic activation by S9 mix with negative outcome performed on 4 Salmonella strains.

Respiratory sensitisation

Endpoint conclusion

Additional information:

No data available.

Justification for classification or non-classification

The available information does not point to a skin sensitization potential in vivo. No protein binding alert was obtained via in silico assessment with the OECD Toolbox. The substance was tested in chemico for the first key event of skin sensitization (peptide reactivity) and in vitro for the second key event (keratinocyte activation). Both tests revealed negative results. A third test (activation of dendritic cells) is ongoing.

The above listed tests and procedures are adequate for non-classification regarding skin sensitization and risk assessment according to point 8.3 of Annex VII of Regulation (EC) 1907/2006.