Lithium 2-aminobenzothiazole-6-sulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3-8 April, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted according to OECD guideline No. 442C

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 4 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This test is part of a tiered strategy for skin sensitisation assessment

Test material

Test material information

Constituent 1

Reference substance name:

Lithium 2-aminobenzothiazole-6-sulphonate

EC Number:

265-367-3

EC Name:

Lithium 2-aminobenzothiazole-6-sulphonate

Cas Number:

65072-36-8

Molecular formula:

C7H6N2O3S2.Li

IUPAC Name:

lithium 2-amino-1,3-benzothiazole-6-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Bulk Density (g/ml): 0.39
- Particle size distribution (μm): 19.6
- Water solubility: 20%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: IR2510181
- Expiration date of the lot/batch: 25 October 2019
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: soluble at 100 mM in milli-Q water, 20% soluble in water

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in milli-Q water
- Preliminary purification step (if any): Not specified
- Final dilution of a dissolved solid, stock liquid or gel: 100 mM

FORM AS APPLIED IN THE TEST
- 2-ABT lithium sulphonate (off-white powder) was dissolved in milli-Q water for testing

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
2-ABT lithium sulphonate was dissolved in milli-Q water at 100 mM. 50 µL of this solution was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in sodium phosphate buffer at pH 7.5) and 200 µL of acetonitrile. In parallel, 250 µL of the 2-ABT lithium sulphonate solution was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2). The samples were incubated for 24 (± 2) hours at 25°C and protected from light, after which they underwent a visual inspection. Samples presenting precipitate and phase separation (micelles) were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected into the HPLC/UV system. Otherwise, the samples were directly injected into the HPLC/UV system. The respective chromatograms were then analysed and interpreted for the cysteine/lysine reactivity potential of 2-ABT lithium sulphonate.

Results and discussion

Positive control results:

Cinnamaldehyde (99.5% purity), a known skin sensitiser, was dissolved in acetonitrile at 100 mM. 50 µL of the solution was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in sodium phosphate buffer at pH 7.5) and 200 µL of acetonitrile. In parallel, 250 µL of cinnamaldehyde solution was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2). Following HPLC/UV analysis, the average % depletion of cysteine peptide and lysine peptide was 72.15 (SD = 0.18) and 59.91 (SD = 0.50), respectively. The mean depletion rate (%) of cinnamaldehyde was 66.03 (high reactivity). These data are within the ranges of the OECD test guideline (442C) acceptance criteria (60.8-100% and 40.2-69.0% for the cysteine and lysine peptide, respectively), therefore cinnamaldehyde was identified by the laboratory as a viable positive control.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Mean score from samples 1, 2 and 3 of 2-ABT lithium sulphonate

Parameter:

other: Cysteine peptide % depletion

Value:

22.76

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Low reactivity

Remarks:

based on the cysteine 1:10-only prediction model. However, precipitates were observed in the cysteine sample following incubation, so the peptide depletion may be underestimated.

Results 2

Run / experiment:

other: Mean score from samples 1, 2 and 3 of 2-ABT lithium sulphonate

Parameter:

other: Lysine peptide % depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Value set to 0 due to negative depletion

Remarks:

The test item co-eluted with the lysine peptide. Since the interference percentage of this lysine co-elution was less than 10% of the mean peptide-peak area of the reference control C (i.e. 1.6%), co-elution was considered as baseline noise and integration of the lysine peak was allowed.

Results 3

Run / experiment:

other: Mean score from samples 1, 2 and 3 of 2-ABT lithium sulphonate

Parameter:

other: Mean depletion rate (%)

Value:

11.38

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Low reactivity - However, since precipitates were observed at the end of the incubation with the cysteine peptide, the peptide depletion may be underestimated.

Applicant's summary and conclusion

Interpretation of results:

other: Indication of positive skin sensitising effect.

Conclusions:

In an in chemico skin sensitisation test (DPRA), conducted according to OECD test guideline 442C and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was determined to have the potential to cause skin sensitisation.

Executive summary:

In a study, conducted according to OECD test guideline 442C and to GLP, 100 mM lithium 2-aminobenzothiazole-6-sulphonate (in water) was added to peptide solutions containing cysteine or lysine to assess its potential to cause skin sensitsation.

The samples were incubated for 24 (± 2) hours at 25°C and protected from light, after which they underwent a visual inspection. Samples presenting precipitate and phase separation were centrifuged at 400g for 5 minutes and the supernatant was injected into the HPLC/UV system. Otherwise, the samples were directly injected into the HPLC/UV system. The respective chromatograms were then analysed and interpreted for the cysteine/lysine reactivity potential of lithium 2-aminobenzothiazole-6-sulphonate. The mean percentage cysteine and lysine depletion was 11.38%, however, since precipitates were observed in the cysteine sample following incubation, the peptide depletion may be underestimated.

In this reliable study, lithium 2-aminobenzothiazole-6-sulphonate was determined to have the potential to cause skin sensitisation.