Lithium 2-aminobenzothiazole-6-sulphonate

Endpoint summary

Description of key information

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.

In an in vitro test using isolated chicken eyes, conducted according to OECD test guideline 438 and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was determined to be non-irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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

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

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)

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)

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment 1: Conducted on 10 January, 2019
Experiment 2: Conducted on 15 January, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted: 25th June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Physiological saline was used to rinse the eyes after the addition of 2-ABT lithium sulphonate. 30 mg 2-ABT lithium sulphonate was found to dissolve in 1 mL physiological saline.
- 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)

Species:

chicken

Strain:

other: ROSS 308

Remarks:

Chicken for human consumption

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
- Number of animals: Not specified
- Characteristics of donor animals: Approximately 7 weeks old
- Storage, temperature and transport conditions of ocular tissue: Chicken heads were transported at an ambient temperature and at the earliest convenience. Upon arrival, the heads were inspected for appropriate quality, wrapped with tissue paper moistened with saline and then placed in a closed plastic box (4-5 heads per box)
- Time interval prior to initiating testing: Not specified, but the heads were processed within 2 hours of their arrival at Citoxlab Hungary Ltd. in each experiment
- Indication of any existing defects or lesions in ocular tissue samples: Not specified
- Indication of any antibiotics used: Not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg

Duration of treatment / exposure:

10 seconds
Observation period: 240 ± 5 minutes

Number of animals or in vitro replicates:

Overall, 6 eyes (3 in each test) were treated with 2-ABT lithium sulphonate
Overall, 6 eyes (3 in each test) were treated with imidazole (positive control)
Overall, 2 eyes (1 in each test) were treated with physiological saline (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

Chicken eyelids were cut away with scissors, avoiding damaging the cornea. A drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.

If the cornea was in good condition, the eyeball was removed from the orbit, without cutting the optical nerve too short and avoiding pressure on the eye, by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eyes were then placed in steel clamps, with the cornea positioned vertically and the eye in the correct relative position. The clamp with the eyeball was transferred to the chamber of the superfusion apparatus and positioned in a way that ensured that the entire cornea was supplied with physiological saline solution, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minute. The chamber door was closed, except for manipulations and examination, to maintain temperature and humidity.

Eyes were removed from the superfusion apparatus and were examined again with the slit lamp microscope to ensure that they were in good condition. The focus of the microscope was adjusted to ensure that physiological saline was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured and any eye with a cornea thickness deviating more than 10% from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The temperature of the chambers of the superfusion apparatus were controlled (32±1.5°C) during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. No changes in thickness (0.0%) were observed in the eyes in Experiment I and no significant changes in thickness (1.6%) were observed in the eyes in Experiment II. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES

The irritating effects of 2-ABT lithium sulphonate were assessed using 6 chicken eyes (two experiments, using 3 eyes each)

NEGATIVE CONTROL USED

Physiological saline (30 µL)

POSITIVE CONTROL USED

Powdered imidazole (30 mg)

APPLICATION DOSE AND EXPOSURE TIME

30 mg 2-ABT lithium sulphonate was applied to the chicken eye for 10 seconds

OBSERVATION PERIOD
240 ± 5 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual 2-ABT lithium sulphonate if possible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal swelling and opacity were measured in both control and test eyes at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse and pre-treatment. Minor variations within approximately ± 5 minutes were considered acceptable.
- A slit-lamp microscope (Haag-Streit Bern 900) was used to measure fluorescein retention at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.

SCORING SYSTEM:

- Mean corneal swelling (%):
0 to 5 = ICE Class I
>5 to 12 = ICE Class II
>12 to 18 ( >75 min after treatment ) = ICE Class II
>12 to 18 ( ≤75 min after treatment ) = ICE Class III
>18 to 26 = ICE Class III
>26 to 32 ( >75 min after treatment ) = ICE Class III
>26 to 32 ( ≤75 min after treatment ) = ICE Class IV
>32 = ICE Class IV

- Mean maximum corneal opacity score
0.0 – 0.5 = ICE Class I
0.6 – 1.5 = ICE Class II
1.6 – 2.5 = ICE Class III
2.6 – 4.0 = ICE Class IV

- Mean fluorescein retention score at 30 minutes post-treatment
0.0 – 0.5 = ICE Class I
0.6 – 1.5 = ICE Class II
1.6 – 2.5 = ICE Class III
2.6 – 3.0 = ICE Class IV

DECISION CRITERIA: Conclusions of each test were based on the OECD guideline quantitative assessments. 2-ABT lithium sulphonate was classified according to the EC and GHS.

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling

Run / experiment:

Experiment 1 - observation at 240 minutes

Value:

1.1

Negative controls validity:

valid

Remarks:

In experiments one and two, the mean maximum corneal swelling values were 0.0% with physiological saline

Positive controls validity:

valid

Remarks:

In experiments one and two, the respective mean maximum corneal swelling values were 27.7 and 27.0% at 240 minutes with imidazole

Remarks on result:

no indication of irritation

Remarks:

(ICE Class I)

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling

Run / experiment:

Experiment 2

Value:

4.3

Negative controls validity:

valid

Remarks:

In experiments one and two, the mean maximum corneal swelling values were 0.0% with physiological saline

Positive controls validity:

valid

Remarks:

In experiments one and two, the respective mean maximum corneal swelling values were 27.7 and 27.0% at 240 minutes with imidazole

Remarks on result:

no indication of irritation

Remarks:

(ICE Class I)

Irritation parameter:

cornea opacity score

Remarks:

Mean maximum corneal opacity

Run / experiment:

Experiment 1

Value:

1

Negative controls validity:

valid

Remarks:

In experiments one and two, the mean maximum corneal opacity scores were 0 (maximum 4) with physiological saline

Positive controls validity:

valid

Remarks:

In experiments one and two, the mean maximum corneal opacity scores were 4 (maximum 4) with imidazole

Remarks on result:

other: Slight corneal opacity (severity 1, ICE Class II) was observed in all three eyes

Irritation parameter:

cornea opacity score

Remarks:

Mean maximum corneal opacity

Run / experiment:

Experiment 2

Value:

1

Negative controls validity:

valid

Remarks:

In experiments one and two, the mean maximum corneal opacity scores were 0 (maximum 4) with physiological saline

Positive controls validity:

valid

Remarks:

In experiments one and two, the mean maximum corneal opacity scores were 4 (maximum 4) with imidazole

Remarks on result:

other: Slight corneal opacity (severity 1, ICE Class II) was observed in all three eyes

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

Experiment 1

Value:

1

Negative controls validity:

valid

Remarks:

In experiments one and two, the mean fluorescein retention scores were 0 (maximum 3) with physiological saline

Positive controls validity:

valid

Remarks:

In experiments one and two, the mean mean fluorescein retention scores were 3 (maximum 3) with imidazole

Remarks on result:

other: Slight fluorescein retention (severity 1, ICE Class II) was seen in all three eyes

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

Experiment 2

Value:

0.83

Negative controls validity:

valid

Remarks:

In experiments one and two, the mean fluorescein retention scores were 0 (maximum 3) with physiological saline

Positive controls validity:

valid

Remarks:

In experiments one and two, the mean mean fluorescein retention scores were 3 (maximum 3) with imidazole

Remarks on result:

other: Slight fluorescein retention (severity 0.5 in one eye and severity 1 in two eyes, ICE Class II) was seen in all three eyes

Other effects / acceptance of results:

In experiments 1 and 2, imidazole (positive control) was stuck on all cornea surfaces (6/6 eyes) after the post-treatment rinse and had not cleared when observed 240 minutes later.

Interpretation of results:

GHS criteria not met

Remarks:

1 x ICE Class I and 2 x ICE Class II in both experiments

Conclusions:

In an in vitro test using isolated chicken eyes, conducted according to OECD test guideline 438 and to GLP, lithium 2-aminobenzothiazole-6-sulphonate was determined to be non-irritating to the eyes.

Executive summary:

In an in vitro eye irritation study, conducted according to OECD test guideline 438 and to GLP, 30 mg of lithium 2-aminobenzothiazole-6-sulphonate was applied to six isolated chicken eyes (3/experiment) for 10 seconds. The eyes were then rinsed using physiological saline and evaluated for alterations in corneal thickness and opacity (up to four hours post-rinsing) and fluorescein retention (30 minutes post-rinsing).

There was no significant swelling of the cornea (mean ≤5%) during the observation period in either experiment, however, slight corneal opacity (severity 1) was observed in all eyes. Additionally, severity 1 (slight) fluorescein retention was noted in 5/6 eyes, with the other displaying a severity score of 0.5. Therefore, lithium 2-aminobenzothiazole-6-sulphonate was assigned an overall ICE Class of 1xI (mean maximum corneal swelling) and 2xII (mean maximum cornea opacity and mean fluorescein retention).

In this reliable in vitro study, lithium 2-aminobenzothiazole-6-sulphonate was determined to be non-irritating to isolated chicken eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Does not warrant classification according to EU CLP criteria.

In the in vitro skin corrosivity test, the mean cell viability was > 35% (97.6%), compared to the negative control, after 240 minutes of exposure, therefore lithium 2-aminobenzothiazole-6-sulphonate is classified as being non-corrosive to the skin. In the in vitro skin irritation test, the mean cell viability was > 50% (91.8%), compared to the negative control, after 15 minutes of exposure and 42 hours post incubation, therefore lithium 2-aminobenzothiazole-6-sulphonate is classified as being non-irritating to the skin.

In the in vitro eye irritation test, lithium 2-aminobenzothiazole-6-sulphonate was assigned an overall ICE Class of 1xI (mean maximum corneal swelling) and 2xII (mean maximum corneal opacity and mean fluorescein retention). Therefore, lithium 2-aminobenzothiazole-6-sulphonate is classified as being non-irritating to the eyes.