Lithium 2-aminobenzothiazole-6-sulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion, other

Remarks:

In vitro skin corrosion and skin irritation (combined) study.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9-11 January, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted: 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted: 28 July 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 Bis (In vitro skin corrosion: Reconstructed Human Epidermis Test Method)

Version / remarks:

No 440/2008, Annex Part B, Dated May 31st, 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

No. 640/2012, Annex III, Dated 6 July, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Controlled room temperature (15-25 °C, ≤70% relative
humidity)
- 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: 2-ABT lithium sulphonate was ground to a fine powder
- Preliminary purification step: Not specified

FORM AS APPLIED IN THE TEST
- 2-ABT lithium sulphonate was applied in its original form (although it was ground to a fine powder)

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™ (SM) model

Source species:

human

Cell type:

other: Normal human keratinocytes

Cell source:

other: Adult donors

Source strain:

other: Not applicable

Details on animal used as source of test system:

The donors blood verified:
- the absence of HIV1 and 2 antibodies
- the absence of hepatitis C antibodies
- the absence of hepatitis B antigen HBs
The donors epidermal cells verified:
- the absence of bacteria, fungus and mycoplasma

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity and irritation testing in
an international validation study and its use is recommended by the relevant OECD
guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439);
therefore, it was considered to be suitable for this study.

Vehicle:

other: Irritation test: 10 μl of distilled water was applied to the epidermal surface before the addition of 10 mg 2-ABT lithium sulphonate

Remarks:

Corrosivity test: 100 μl of physiological saline was added to 2-ABT lithium sulphonate after it had been applied to the epidermal surface

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM)
- Tissue batch number(s): 19-EKIN-001
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 9 January, 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
Irritation test: 24.0-24.9 °C
Corrosivity test: 23.2-24.0 °C
- Temperature of post-treatment incubation:
Irritation test: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 washing step using phoshpate buffered saline (volume not specified)
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: Not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT working solution
- Incubation time: 3 hours
- Spectrophotometer: Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 13 August 2018, calibration is valid until August 2020
- Wavelength: 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Irritation test: phosphate buffered saline optical density = 0.784
Corrosivity test: physiological saline optical density = 0.849
- Barrier function:
IC50 (SDS concentration, MTT test) = 2.3 mg/mL
- Morphology:
Histology scoring (HES stained vertical paraffin sections) - result: 23.3 ± 0.3 (CV = 1.2%)
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum - result: satisfactory
- Contamination:
Blood:
absence of HIV1 and 2 antibodies
absence of hepatitis C antibodies
absence of hepatitis B antigen HBs
Epidermis:
absence of bacteria, fungus and mycoplasma
- Reproducibility: Not specified

NUMBER OF REPLICATE TISSUES:
- Irritation test: three replicate epidermis units
- Corrosivity test: two replicate epidermis units

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Living tissue - Reconstructed human epidermis (EPISKIN™(SM) model)
- No. of replicates : Four (irritation test: 2, corrosivity test: 2)
- Method of calculation used: Optical density measured at 570 nm

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the mean cell viability for both skin units is greater than or equal to 35% of the negative control after 4 hours of exposure
- The test substance is considered to be non-irritating to skin if the mean cell viability for all three skin units is greater than 50% of the mean viability of the negative controls after 15 minutes of exposure and 42 hours post-incubation

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: yes, MTT control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied:
Irritation test: 10 mg
Corrosivity test: 20 mg

NEGATIVE CONTROL
- Amount(s) applied:
Irritation test: 50 µL of phosphate buffered saline
Corrosivity test: 50 µL of physiological saline (0.9% (w/v) NaCl solution)

POSITIVE CONTROL
- Amount(s) applied:
Irritation test: 50 µL of 5% (w/v) sodium dodecyl sulphate solution
Corrosivity test: 50 µL glacial acetic acid

Duration of treatment / exposure:

Irritation test: 15 minutes
Corrosivity test: 4 hours

Duration of post-treatment incubation (if applicable):

Irritation test: 42 hours (±1 hour)

Number of replicates:

Irritation test: 3 reconstructed epidermis units (area: 0.38 cm²) each for 2-ABT lithium sulphonate, the positive control and the negative control
Corrosivity test: 2 reconstructed epidermis units (area: 0.38 cm²) each for 2-ABT lithium sulphonate, the positive control and the negative control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: % mean cell viability

Run / experiment:

Irritation testing: 10 mg 2-ABT lithium sulphonate was applied 3 reconstructed epidermis units for 15 minutes at room temperature (24.0-24.9°C).

Value:

91.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% mean cell viability obtained with phosphate buffered saline (50 μl)

Positive controls validity:

valid

Remarks:

5.3% mean cell viability obtained with 5% (w/v) sodium dodecyl sulphate solution (50 μl)

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: % mean cell viability

Run / experiment:

Corrosivity testing: 20 mg 2-ABT lithium sulphonate was applied to 2 reconstructed epidermis units for 4 hours at room temperature (23.2-24.0°C).

Value:

97.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% mean cell viability obtained with 50 μl physiological saline (0.9% (w/v) NaCl)

Positive controls validity:

valid

Remarks:

1.1% mean cell viability obtained with glacial acetic acid (50 μl)

Remarks on result:

other: no indication of corrosivity

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro reconstructed human epidermis (EPISKIN™ (SM) model) test, conducted according to OECD test guidelines 431 and 439 and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was determined to be non-corrosive and non-irritating to the skin.

Executive summary:

In an in vitro study, conducted according to OECD test guidelines 431 and 439 and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was applied to an EPISKIN™ (SM) reconstructed human epidermis model to assess its potential to cause skin irritation and/or corrosion.

In the irritation study, three reconstructed epidermis units were exposed to 10 mg lithium 2-aminobenzothiazole-6-sulphonate for 15 minutes at room temperature. The units were then rinsed using phosphate buffered saline and incubated at 37 °C for 42 hours. In the corrosivity study, two reconstructed epidermis units were exposed to 20 mg lithium 2-aminobenzothiazole-6-sulphonate for 4 hours at room temperature and then rinsed using phosphate buffered saline.

In both tests, MTT (2 mL of 0.3 mg/mL MTT working solution) was added to each epidermis unit and the units were incubated at 37 °C for 3 hours. The mean cell viability was calculated by assessing the optical density of the samples using a plate reader at 570 nm. In the irritation and corrosivity tests, the mean cell viability was calculated to be 91.8 and 97.6%, respectively.

In this reliable in vitro study, lithium 2-aminobenzothiazole-6-sulphonate was determined to be non-corrosive and non-irritating to human reconstructed epidermis.