Disodium naphthalene-1,5-disulphonate

Endpoint summary

Description of key information

The available information obtained in silico, in chemico and in vitro does not point to a potential skin sensitizing activity 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, OECD 442C) and in vitro for the second (keratinocyte activation, OECD 442D) and third key event of skin sensitization (dendritic cell activation, OECD 442E). All tests revealed negative results.

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

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CHNHFC1602
Disodium-naphthalene-1,5-disulphonate was tested as monohydrate in this test. Calculations of all test article concentrations stated in this report thus include a correction for content (purity) using a factor of 1.056. The correction factor compensates for the crystallisation
water content of the test compound, so that the results are also reliable for Di-sodium-naphthalene-1,5-disulfonic acid CAS: 1655-29-4).

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, 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 prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).

The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5ºC. Relative peptide concentration is measured by reversed phase (C18) high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. The synthetic peptides contain phenylalanine to aid in the detection.
The test item was dissolved and tested according to the given test procedure. Cinnamic aldehyde was used as positive control at a concentration of 100 mmol/L in acetonitrile.

Cysteine and lysine peptide solutions were incubated at 1:10 and 1:50 ratio in glass auto sampler vials with the test item solution for 24 hours at 25±2.5°C in the dark. Samples were visually inspected for precipitation or phase separation before HPLC analysis. The test item was analyzed in triplicate for both peptides. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. HPLC analysis for the cysteine and lysine peptides were performed on separate days with the test item solutions freshly prepared for both assays on each day. The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards. Percent peptide depletion is calculated according to OECD Guideline.

The following criteria should be met for a test chemical’s results to be considered valid: a) the maximum standard deviation for the test chemical replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion, b) the mean peptide concentration of three injections of the reference control C in the appropriate solvent should be 0.50±0.05 mM.

According to the study protocol the mean percent cysteine and percent lysine depletion value was calculated for the test item and the positive control. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion can be used to support the discrimination between skin sensitizers and non-sensitizers in the framework of an IATA.

Positive control results:

The positive control cinnamic aldehyde led to a depletion of 71.49% cysteine peptide and 61.72% lysine peptide. The mean cysteine/lysine peptide depletion was calculated with 66.61% leading to a positive results (high reactivity class according to the cysteine 1:10/lysine 1:50 prediction model).

Run / experiment:

other: mean of 2 runs

Parameter:

other: % depletion in the cysteine 1:10/lysine 1:50 prediction model:

Value:

1.08

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA prediction: negative

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean % cysteine peptide depletion

Value:

2.16

Vehicle controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean % lysine depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes

The test item visually appeared a clear solution in water at the test concentration of 100 mmol/L. No precipitation occured before or after incubation.
Absorbance at 220 nm was not observed. Retention time similar to peptide was not observed. Co-elution of the test item with the peptides was not observed.

Results of the DPRA:

Test item	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	reactivity class	DPRA prediction
positive control (DNCB)	71.49	61.72	66.61	high	positive
test item	2.16	0	1.08	minimal	negative

Executive summary:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, 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 prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Since the test item visually appeared a clear solution in water at the test concentration of 100 mmol/L this solvent was used in the DPRA. No precipitaton of the test item occured before or after incubation. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (1.08%). According to the prediction model “minimal reactivity” was derived for the test item in water, leading to a DPRA prediction of “negative“.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

of February 2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CHNHFC1602
Disodium-naphthalene-1,5-disulphonate was tested as monohydrate in this test. Calculations of all test article concentrations stated in this report thus include a correction for content (purity) using a factor of 1.056. The correction factor compensates for the crystallisation
water content of the test compound, so that the results are also reliable for Di-sodium-naphthalene-1,5-disulfonic acid CAS: 1655-29-4).

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised 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.

Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D (batch number 42, passage 12 for Experiment 1 and batch number 45, passage 12 for Experiment 2).

Preparation of Cultures:
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 9% foetal bovine serum and 1% Geneticin.

Treatment:
In 96-well plates, incubated at 37±1°C, 5% (v/v) CO2, for 48±1 hours in medium with serum but without Geneticin. For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.

Cytotoxicity Assessment:
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements:
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive controls:
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

Negative controls:
The negative control was diluted into culture medium containing serum so that the final concentration was 1%. An aqueous solvent was used, therefore DMSO was added to the treatment solutions at 1% (final concentration).

Positive control results:

The assay aceptance criteria for the positive controls were met:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiments 1 and 2.
The EC1.5 values for the positive control were 8.51 and 8.50 µM in Experiments 1 and 2, respectively. The average induction for the positive control at 64 µM was 4.97 in Experiment 1 and 17.47 in Experiment 2. Although the latter value was above the range specified in the protocol, the assay was considered valid as there was a clear dose response with increasing luciferase activity induction.

Run / experiment:

other: experiment 1

Parameter:

other: Imax - Exp. 1 -

Value:

1.19

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: experiment 2

Parameter:

other: Imax - Exp. 2 -

Value:

1.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: proven

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 9.44% and 15.84% in Experiments 1 and 2, respectively.

All assay acceptance criteria were met.

Since the Imax in both experiments was less than 1.5-fold, the test article was considered to be negative in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed acording to OECD 442D. The test article was dissolved in water to the final concentration of the stock solution (200 mM) with further dilution in water and cell culture medium to obtain final concentrations of 0.98 to 2000 µM. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test. Treatment of the cell for 48 hours with the test item led to the following results: The maximal average fold increases (Imax) were 1.19 and 1.10 for experiments 1 and 2, respectively. There were no EC1.5 values for Experiments 1 or 2 determinable as there were no statistically significant increases in induction. In Experiments 1 and 2, there was no apparent overall dose response for luciferase and the dose response curves were not biphasic.

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

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT))

Version / remarks:

adopted 09 October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CHNHFC1602
Disodium-naphthalene-1,5-disulphonate was tested as monohydrate in this test. Calculations of all test article concentrations stated in this report thus include a correction for content (purity) using a factor of 1.056. The correction factor compensates for the crystallisation
water content of the test compound, so that the results are also reliable for Di-sodium-naphthalene-1,5-disulfonic acid CAS: 1655-29-4).

Details on the study design:

The test item was examined for sensitising properties in the human Cell Line Activation test (h-CLAT). The h-CLAT is an in vitro assay proposed to address the third key event of the skin sensitisation AOP (Adverse Outcome Pathway) by quantifying changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was 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 the solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.
The h-CLAT has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The test article was insoluble in DMSO, but was soluble in saline at 142 mg/mL. Hence, saline was used as vehicle.
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value is used to determine the concentration of test item for the CD86/CD54 expression measurement. In this preliminary experiment (consisting of two independent runs) no effect on viability was noted up to the maximum attainable concentration of 142 mg/mL in saline. Eight dilutions (Eight concentrations) were prepared, by two-fold serial dilution with medium and a final range of concentrations in the plate of 369 to 1420 μg/mL culture medium were used for the expression measurements. DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.
Following cell staining with fluorochrome-tagged antibodies the expression of CD86 and CD54 is analysed via flow cytometry with the aquisition chanel FL-1. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test item-treated cells were calculated compared to the solvent control. The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) is also calculated.

Positive control results:

RFI values for positive control with regard to CD86: 139 and 419 (in Experiment 1, the RFI for CD86 with the positive control was just below 150%. However, as cell viability was greater than 90% at each concentration of positive control and there were no border line responses with the test article, it was considered that the experiment was valid.
RFI values for positive control with regard to CD54: 498 and 979

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity (RFI) for CD86 - Exp. 1

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity (RFI) for CD86 - Exp. 2

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: relative fluorescent intensity (RFI) for CD54 - Exp. 1

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity (RFI) for CD54 - Exp. 2

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The cell viability of the solvent controls (medium) was higher than 90% in each independent run.
In the solvent control (medium), RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For solvent control (medium), the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control in Experiment 1, the RFI for CD86 was just below 150% (i.e. 139%). However, as cell viability was greater than 90% at each concentration of positive control and there were no border line responses with the test article, it was considered that the experiment was valid
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Relative fluorescence intensity (RFI) values:

Concentration (µg/mL)	RFI CD86 - 1st Exp.	RFI CD86 - 2nd Exp.	RFI CD54 - 1st Exp.	RFI CD54 - 2nd Exp.
369.4	86	91	107	101
475.7	78	112	99	99
570.8	86	107	98	101
685.0	93	112	98	90
822.0	82	125	97	100
986.4	89	120	106	104
1183.7	60	110	99	111
1420.5	69	93	121	118
sovent control (medium)	100	100	100	100
positive control (DNCB)	139	419	498	979

Conclusions:

The test article was considered to be negative in the human Cell Line Activation Test at up to and including the maximum attainable concentration.

Executive summary:

The study was conducted to investigate the potential of Di-sodium-naphthalene-1,5 - disulfonic acid monohydrate to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

The test article was insoluble in DMSO, but was soluble in saline at 142 mg/mL. Dose finding assays were conducted to determine a concentration showing 75% THP-1 cell survival (CV75) compared to the solvent control. No effect on viability was noted up to the maximum attainable concentration. In the CD86/CD54 expression experiments 1 and 2 the relative fluorescence intensity (RFI) values of the test article were <150% for CD86 and <200% for CD54 at all tested concentrations compared to the solvent controls. The test article therefore gave a negative prediction in the assay at up to and including the maximum attainable concentration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

An assessment of potential skin sensitization properties based on defined approaches and individual sources was conducted for disodium-naphthalene-1,5-disulphonate within an IATA. The outcome of this assessment is given below:

No animal studies are available for the substance to assess the endpoint 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 dermal bioavailability:

No information is available on dermal bioavailability of the substance. 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. Based on these physical-chemical parameters 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; Schlecker 2017):

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, Schaub 2018):

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)). 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, Dreher 2018):

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). 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).

Since the Imax in both experiments was below 1.5-fold, disodium-naphthalene-1,5-disulphonate monohydrate 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, Dreher 2018):

Disodium-naphthalene-1,5-disulphonate was tested as monohydrate  in the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test following OECD TG 442E (In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT)). 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.

For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (positive or negative). An h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative:

• The relative fluorescence intensity (RFI) of CD86 is equal to or greater than 150% at any tested concentration (with cell viability ≥ 50%);

• The RFI of CD54 is equal to or greater than 200% at any tested concentration (with cell viability ≥ 50%).

Based on the above conditions, if the first two runs give concordant result for CD86 and/or CD54, the h-CLAT prediction is clear (positive or negative) and a third run does not need to be conducted.

Disodium-naphthalene-1,5-disulphonate monohydrate was shown to be insoluble in DMSO but soluble in saline at 142 mg/mL. Dose finding assays were conducted to determine a concentration showing 75% THP-1 cell survival (CV75) compared to the solvent control. No effect on viability was noted at up to and including the maximum attainable concentration. In the CD86/CD54 expression experiments 1 and 2 the relative fluorescence intensity (RFI) values of the test article were <150% for CD86 and <200% for CD54 at all tested concentrations compared to the solvent controls. The test article therefore gave a negative prediction in the assay at up to and including the maximum attainable concentration.

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.

Discussion and conclusion:

This assessment of potential skin sensitizing properties is based on defined approaches and individual sources to be used within an Integrated Approach to Testing and Assessment (IATA). Information generally considered for such an assessment are exposure considerations, available animal and human data, physico-chemical properties, dermal bioavailability, in silico-, in chemico-, and in vitro-tests, and further toxicological data. Experimental in chemico and in vitro studies but no in vivo tests have been performed for this assessment.

No irritating or sensitizing effects to human skin were reported in occupational settings.

No animal tests are available for the substance to assess the endpoint skin sensitization. Based on the physico-chemical properties of the substance (molecular weight, high water solubility, very low log Kow) dermal absorption can be expected to be low. Additionally, no skin irritating potential was recorded in vitro on reconstructed human skin (Leidenfrost, 2017) and in vivo on rabbit skin (Maertins, 1989). No eye irritating effects were recorded in vitro (Leidenfrost, 2017; Rauh, 2017) and in vivo (Maertins, 1989). The substance was negative and thus showed no electrophilicity in an Ames test in vitro (Herbold, 1989).

No skin sensitization alerts were identified in silico by the OECD QSAR Toolbox (Schlecker, 2017).

As in chemico tool the Direct Peptide Reactivity Assay (DPRA, OECD 442C) was used (Schaub, 2018). The DPRA addresses the first key event of skin sensitization, the covalent binding to skin proteins (haptenation). The test substance showed a negative result (‘minimal reactivity’) in the DPRA with a cysteine/lysine peptide depletion of 1.08%. Thus, no binding affinity to proteins became obvious.

The substance was tested for the second key event of skin sensitization, the activation of keratinocytes in vitro, in the KeratinoSens assay (ARE-Nrf2 Luciferase Test, OECD 442D). The test substance was considered negative in this test (Dreher, 2018).

With regard to the third key event of skin sensitization (activation of dendritic cells) the human Cell Line Activation Test (h-CLAT, OECD 442E) was performed.  The test substance gave a negative prediction in the assay at up to and including the maximum attainable concentration (Dreher, 2018).

In conclusion and in applying the Weight of Evidence approach, the available data in this evaluation do 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 (keratinocyte activation) and third key event of skin sensitization (dendritic cell activation). All tests revealed consistently negative results.

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.

Respiratory sensitisation

Endpoint conclusion

Additional information:

No data available.

Justification for classification or non-classification

The available data do 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, OECD 442C) and in vitro for the second (keratinocyte activation, OECD 442D) and third key event of skin sensitization (dendritic cell activation, OECD 442E). All tests revealed consistently negative results. An assessment of skin sensitization based on defined approaches and individual sources was conducted within an IATA.

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.