1,2-Benzisothiazole, 3-methyl-, 1,1-dioxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-01-18 to 2017-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

29 June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1λ⁶,2-benzothiazole-1,1-dione

EC Number:

812-907-6

Cas Number:

34989-82-7

Molecular formula:

C8H7NO2S

IUPAC Name:

3-methyl-1λ⁶,2-benzothiazole-1,1-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular™ (OCL-200-EIT), Lot: 23759, Expiry date: 19 January 2017, Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma, resulting in "not detected". The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3 % (v/v) Triton X-100).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amountapplied: 50 mg

Duration of treatment / exposure:

6 hours (± 15 min) at 37 ± 1 °C

Duration of post- treatment incubation (in vitro):

Post-Soak: 25 ± 2 minute at room temperature
Post-treatment Incubation: 18 hours ± 15 minutes at 37 ± 1 °C

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT), Lot: 23759, Expiry date: 19 January 2017, Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used: see above
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: see above
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The test item showed no direct interaction with MTT. Using of additional control was not necessary.
- Number of tissue replicates used per test chemical and controls: see above
- Wavelength used for quantifying MTT formazan, and measuring device: 96-well plate spectrophotometer at the wavelength of 570 nm
- Description of the method used to quantify MTT formazan: Absorbance / Optical Density of the samples
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 % of the negative control. The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.
- Positive and negative control means and acceptance ranges based on historical data: The mean historical OD value of the negative control tissue was between 1.138 and 2.378. The mean historical OD value of the positive control tissue was between 0.064 and 0.5 with a viability of 3 to 22 %.
- Acceptable variability between tissue replicates for positive and negative controls: The mean OD value of the two negative control tissues should be between 0.8 and 2.5. The acceptable percentage viability for positive control (mean of two tissues) is for 30 minute exposure below 50 % of control viability and for 6 hours exposure below 50 % of control viability.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single chemical is < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals).

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability [%]

Run / experiment:

mean

Value:

68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

15 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: The mean historical OD value of the negative control tissue was between 1.138 and 2.378. The mean historical OD value of the positive control tissue was between 0.064 and 0.5 with a viability of 3 to 22 %.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be not irritating to the eyes in the in vitro Reconstructed human Cornea-like Epithelium.

Executive summary:

The in vitro eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was performed to assess the irritation potential of the test substance. Independent duplicate tissues of EpiOcular were exposed to either the test item, the negative control (deionised water, 50 μL) or the positive control (Methyl acetate, 50 μL) for 6 hours ± 15 minutes. 50 mg of the test item were dispensed directly onto duplicate EpiOcular tissue surface. The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 68 %). Positive (15 %) and negative (100 %) controls showed the expected cell viability values within acceptable limits. All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.