2-[4-(dimethylamino)phenyl]-3,6-dimethylbenzothiazolium chloride

Endpoint summary

Description of key information

Three in chemico studies have been performed with the test item Basic Yellow 1, in order to assess its sensitizing potential.

In the OECD 442C study (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)), that enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine, Basic Yellow 1 showed moderate reactivity towards the cysteine peptide and thus the test item was classified as “sensitizer” in accordance with UN GHS “Category 1”.

The OECD 442D study (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method or in vitro KeratinoSens™ assay) enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitizers and non-sensitizers. In the present study, Basic Yellow 1 did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitizer in accordance with UN GHS category 1.

Finally, the OECD 442E study (In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)) enables detection of the sensitizing potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitizers and non-sensitizers. As the test item is a strong fluorescent test chemical emitting at the same wavelength as FITC, it interferes with the flow cytometric detection. According to the literature, Thioflavine and FITC are measured at very similar wavelengths. Therefore, Basic Yellow 1 cannot correctly be evaluated using FITC conjugated antibodies. Thus, the study was stopped after the first dose finding experiment and neither a result nor a conclusion could be achieved.

Although the OECD 442E study was not possible to be concluded, the results of the other two studies (OECD 442C and OECD 442D) detected a clear sensitizing potential related to the test item. Therefore, Basic Yellow 1 is classified Skin Sens. 1, H317 by Weight of Evidence and conservative approaches. However, no further sub-classification in category 1A or 1B was possible for this endpoint.

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))

Deviations:

no

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay

Run / experiment:

other: Cysteine Peptide Run

Parameter:

other: Mean Peptide Depletion

Value:

29.1

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Lysine Peptide Run

Parameter:

other: Mean Peptide Depletion

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

The samples containing test item and lysine peptide had become solid after the incubation time. Therefore, no measurement was possible and no results concerning the lysine peptide depletion of the test item are reported

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed moderate reactivity towards the cysteine peptide. The test item can be classified as “sensitiser” in accordance with UN GHS “Category 1”.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study Basic Yellow 1 was dissolved in water.

Based on a molecular weight of 318.86 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

The samples containing test item and lysine peptide had become solid after the incubation time. Therefore, no measurement was possible and no results concerning the lysine peptide depletion of the test item are reported.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis the cysteine peptide samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for standard 1 and the positive control samples. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant.

No co-elution of test item with the cysteine peptide peak was observed. Since the lysine peptide samples could not be measured, sensitising potential of the test item was predicted only from the peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was > 13.89% (29.15%). Based on the prediction model 2 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.92%.

8.2. Conclusion

In this study under the given conditions the test item showed moderate reactivity towards the cysteine peptide. The test item can be classified as “sensitiser” in accordance with UN GHS “Category 1”.

The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

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:

other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016

GLP compliance:

yes

Type of study:

activation of dendritic cells

Parameter:

other:

Remarks on result:

not determinable because of methodological limitations

Conclusions:

The test item is a strong fluorescent test chemical emitting at the same wavelength as FITC and interferes with the flow cytometric detection. According to the literature, Thioflavine and FITC are measured at very similar wavelengths.
The test item can therefore not correctly be evaluated using FITC conjugated antibodies. Thus, the study was stopped after the first dose finding experiment.

Executive summary:

Thein vitrohuman cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study Basic Yellow 1 was dissolved in cell culture medium. For the dose finding assay stock solutions with concentrations ranging from 40 mg/mL to 0.313 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Hereby, a clear dose effect curve was observed with a maximum cell viability of 73.9% at the lowest test item concentration of 3.13 µg/mL. As no concentration with a cell viability≥75% was observed, no CV75 could be calculated.

Furthermore, the datashowed a strong shift of the fluorescence signal for the test item compared to the medium (see15Appendix). Therefore, it was assumed, that the test item is a strong fluorescent test chemical emitting at the same wavelength as FITC and interferes with the flow cytometric detection. According to the literature, Thioflavine and FITC are measured at very similar wavelengths.

The test item can therefore not correctly be evaluated using FITC conjugated antibodies. Thus, the study was stopped after the first dose finding experiment.

Reference 3

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)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Parameter:

other:

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study Basic Yellow 1 was dissolved in cell culture medium.

Based on a molecular weight of 318.86 g/mol a stock solution of 50 mM was prepared for experiment 1 and experiment 2. In order to verify the first and second experiment, a third experiment with adapted concentrations was performed. For experiment 3 a stock solution of 1.5 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration ranges were tested in the experiments:

Experiment 1 and experiment 2:

500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 and 0.24 μM

Experiment 3:

15, 7.5, 3.75, 1.88, 0.94, 0.47, 0.23, 0.12, 0.06, 0.03, 0.015, 0.007 μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 42.97 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 69%. The lowest tested concentration with a significant luciferase induction >1.5 (4.36) was found to be 3.91 μM. The corresponding cell viability was >70% (62.1%). The calculated EC1.5 was < 1000 μM (2.09 μM).

In the second experiment, a max luciferase activity (Imax) induction of 83.19 was determined at a test item concentration of 31.25 μM. The corresponding cell viability was 104.8%. The lowest tested concentration with a significant luciferase induction >1.5 (2.62) was found to be 3.91 μM. The corresponding cell viability was >70% (110.6%). The calculated EC1.5 was < 1000 μM (2.45 μM).

In the third experiment, a max luciferase activity (Imax) induction of 26.20 was determined at a test item concentration of 15 μM. The corresponding cell viability was 64.5%. The lowest tested concentration with a significant luciferase induction >1.5 (2.42) was found to be 3.75 μM. The corresponding cell viability was >70% (95.5%). The calculated EC1.5 was < 1000 μM (2.71 μM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as sensitiser.

Conclusion

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance Basic Yellow 1 is classified Skin Sens. 1, H317, according to Regulation (EC) n. 1272/2008.