Lithium 2-aminobenzothiazole-6-sulphonate

Endpoint summary

Description of key information

The skin sensitisation potential of lithium 2-aminobenzothiazole-6-sulphonate has been assessed in chemico, in vitro and in vivo.

In chemico, in a study conducted according to OECD test guideline 442C and to GLP, 100 mM of the test substance (in water) was added to peptide solutions containing cysteine or lysine (the Direct Peptide Reactivity Assay). The mean percentage cysteine and lysine depletion was 11.38%, indicative of "low reactivity".

In vitro, lithium 2-aminobenzothiazole-6-sulphonate was tested in two independent runs using KeratinoSens cells (following OECD test guideline 442D). The EC1.5, the concentration calculated to cause a 1.5-fold increase in luciferase gene induction, was 490.7 µM, and the Imax, the maximal induction factor of luciferase activity compared to the negative control, was 2.68. Both values are indicative of the potential to cause skin sensitisation by activating the Nrf2 transcription factor.

However in vivo in the murine LLNA (OECD test guideline 429), lithium 2-aminobenzothiazole-6-sulphonate, when tested at up to the maximum soluble concentration, 25% in 1% Pluronic, gave no indication of a skin sensitising potential. Three concentrations (5%, 10% and 25%) were tested; there was no evidence of a dose response.

Therefore it is concluded that lithium 2-aminobenzothiazole-6-sulphonate is not a skin sensitiser, and no classification or labelling for skin sensitisation is required for this substance.

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:

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

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

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

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.

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.

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.

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.

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16-29 April, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Deviation from the OECD guideline recommended filtration following test item preparation for sterilisation prior to treatment

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: June 2018

Deviations:

yes

Remarks:

The test item formulation was not filtered for sterilisation prior to treatment, based on it being a suspension, rather than a solution. Filtration would have considerably decreased the test item concentration (particles would be retained in the filter).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.

The KeratinoSens™ assay uses an immortalised adherent human keratinocyte cell line (HaCaT cell line), transfected with a selectable plasmid to quantify luciferase gene induction as a measure of activation of Keap1-Nrf2-antioxidant/electrophile response element (ARE)1 and has been validated as a useful in vitro system for assessing the skin sensitising potential of compounds.

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: 20% soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in water
- Preliminary purification step: Not purified by filtration
- Final dilution of a dissolved solid, stock liquid or gel: 200 mM

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

Details on the study design:

2-ABT lithium sulphonate was tested in two independent runs using KeratinoSens cells placed on 96-well plates and grown on culture medium for 24 hours at 37°C. The medium was removed and replaced by 150 µL of medium containing 2-ABT lithium sulphonate at 12 concentrations (range: 0.98-2000 µM). The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and their luciferase production was measured by flash luminescence. In parallel, the cytotoxicity of 2-ABT lithium sulphonate was measured by a MTT reduction test.

Run / experiment:

other: Mean score from run 1 (Imax = 2.94) and run 2 (Imax = 2.42)

Parameter:

other: Imax

Remarks:

Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured

Value:

2.68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Geometric mean from run 1 (EC1.5 = 454.16) and run 2 (EC1.5 = 530.34)

Parameter:

other: EC1.5 (µM)

Remarks:

Extrapolated concentration for a 1.5-fold increase in luciferase gene induction

Value:

490.77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Run 1: noted at concentrations ≥ 500 µM Run 2: noted at concentrations ≥ 1000 µM

Parameter:

other: Gene induction (µM)

Remarks:

Mean induction concentration: the concentration at which 2-ABT lithium sulphonate caused gene inductions above the threshold of 1.5

Value:

500

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Significant gene inductions (in comparison to the negative control) above the threshold of 1.5 were noted at concentrations ≥ 500 µM in the first run and ≥ 1000 µM in the second, with an apparent overall dose response relationship.

Other effects / acceptance of results:

The average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. This was not fulfilled, it was 19.17 and 8.74 in the first and second runs, respectively. However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in each run, this was considered not to have any impact on the validity of the results.

Interpretation of results:

other: Indication of positive skin sensitising effect.

Conclusions:

In an in vitro skin sensitisation assay (KeratinoSens), conducted according to OECD test guideline 442D and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was determined to have the potential to cause skin sensitization by activating the Nrf2 transcription factor.

Executive summary:

In an in vitro study, conducted according to OECD test guideline 442D and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was tested in two independent runs using human-derived KeratinoSens cells to assess its potential to cause skin senistisation.

KeratinoSens cells were placed on 96-well plates and grown on culture medium for 24 hours at 37°C. The medium was removed and replaced by 150 µL of medium containing lithium 2-aminobenzothiazole-6-sulphonate at 12 concentrations (range: 0.98-2000 µM). The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and their luciferase production was measured by flash luminescence. Gene inductions, above the threshold of 1.5, were noted at concentrations ≥ 500 µM in the first run and ≥ 1000 µM in the second, with an apparent overall dose response relationship.

In this reliable in vitro study, lithium 2-aminobenzothiazole-6-sulphonate was determined to have the potential to cause skin sensitisation by activating the Nrf2 transcription factor in KeratinoSens cells.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June - 30 July 2019.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Slight deviation from OECD guideline recommended temperature and humidity ranges - not expected to impact study findings or validity.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

The actual temperature range was 19.7 - 25.3 °C instead of 22 ± 3 °C. The actual relative humidity range was 32 - 77% instead of 30-70% as it was indicated in the Study Plan. All animals (test & control) were subject to the same environmental conditions.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: IR2510181.
- Expiration date of the lot/batch: 25 October 2019.

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida 57, 33049 Italy.
- Females nulliparous and non-pregnant: Yes.
- Microbiological status of animals, when known: SPF at arrival.
- Age at study initiation: 11 weeks.
- Weight at study initiation: 21.6 - 24.6 g (the weight variation in animals in the study did not exceed ±20% of the mean weight).
- Housing: Group caging in Type II polypropylene/polycarbonate cages. Mice were provided with glass tunnel-tubes.
- Diet: Commercial diet provided ad libitum.
- Water: Tap water provided ad libitum.
- Acclimation period: 28 days.
- Indication of any skin lesions: No. Only healthy animals were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 – 25.3°C. This is a deviation from the standard 22 ± 3°C.
- Humidity (%): 32 - 77%. This is a deviation from the standard 30 - 70%.
In both cases, the deviations from target environmental parameters were of relatively short duration. All animals, control and treated, were subject to the same environmental conditions. These differences of the environmental parameters were considered not to adversely affect the results or integrity of the study as confirmed by the clinical Veterinarian.
- Air changes (per hr): 15 - 20.
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours. Light daily from 06.00 to 18.00.

IN-LIFE DATES: Not specified.

Vehicle:

other: 1% aqueous Pluronic® PE9200.

Concentration:

5, 10 and 25% (w/v).

No. of animals per dose:

4.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Maximum test item concentration (25% w/v) was determined in a preliminary test.
- Irritation: No irritation at maximum test item concentration in preliminary test.
- Systemic toxicity: No systemic toxicity at maximum test item concentration in preliminary test.
- Ear thickness measurements: Ear thickness values and ear punch weights taken during preliminary test at maximum test item concentration were within the acceptable range.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA.
- Criteria used to consider a positive response: Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:

During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

On Day 6, each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of tritiated thymidine (3HTdR). Once injected, the mice were left for 5 hours (± 30 minutes).

Five hours (± 30 minutes) after injection of 3HTdR, the mice were euthanized and the draining auricular lymph nodes were excised. The incorporation of 3HTdR in the lymph nodes was measured by scintillation counting.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

The positive control, 25% (w/v) hexylcinnamic acid in 1% Pluronic vehicle, was detected as a true positive, with a SI of 4.7.

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (1% Pluronic only)

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

5% Li 2-ABTS in 1% Pluronic

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

10% Li 2-ABTS in 1% Pluronic

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

25% Li 2-ABTS in 1% Pluronic

Interpretation of results:

GHS criteria not met

Conclusions:

Lithium 2-aminobenzothiazole-6-sulphonate, tested at up to 25% in 1% Pluronic, gave no indication of a skin sensitising potential in the murine LLNA.

Executive summary:

In an OECD guideline study, conducted to GLP, lithium 2-aminobenzothiazole-6-sulphonate, tested at up to the maximum soluble concentration, 25% in 1% Pluronic, gave no indication of a skin sensitising potential when tested in the murine LLNA. Three concentrations (5%, 10% and 25%) were tested; there was no evidence of a dose response. No classification or labelling for skin sensitisation is required for this substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Indications of skin sensitisation potential were seen in the DPRA and KeratinoSens assays. On this basis, an h-CLAT assay was not conducted and, following the ITS for skin sensitisation, an in vivo murine LLNA was carried out. There was no evidence of a dose response across three concentrations and, when tested up to the maximum soluble concentration, lithium 2-aminobenzothiazole-6-sulphonate gave no evidence of a skin sensitising effect in the LLNA. As such, no CLP classification is required for this endpoint.