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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-14 to 2016-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number: 16-EKIN-037
- Expiry date: 2016-09-19
- Date of initiation of testing: 2016-09-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL, 1 step
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control mean: 13 %, min.: 2 %, max.: 39 %

NUMBER OF REPLICATE TISSUES:
3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls and 2 replicates color controls were used.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item showed no direct interaction with MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 μL
- Concentration: 1x

POSITIVE CONTROL
- Amount applied: 10 μL
- Concentration: 5 %

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls and 2 replicates color controls were used.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

143

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

16

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: Non Specific Colour % (NSC %): 2.1

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method the test laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes, each calculated standard deviation value (SD) for the % viability was below 18.

Any other information on results incl. tables

Substance	Replicate	Optical Density (OD)	Viability (%)
Negative Control: 1x PBS	1	1.018	117
	2	0.823	95
	3	0.765	88
	mean	0.869	100
	standard deviation (SD)	15.22
Positive Control: SDS (5 % aq.)	1	0.167	19
	2	0.168	19
	3	0.072	8
	mean	0.136	16
	standard deviation (SD)	6.34
Test Item	1	1.355	156
	2	1.102	127
	3	1.281	147
	mean	1.246	143
	standard deviation (SD)	14.97

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be not irritating in the in vitro human skin model test with EPISKIN model.

Executive summary:

The reconstituted human epidermis in the EPISKIN model study according to OECD 439 was performed to assess the irritation potential of the test substance. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. As the test item has an intrinsic colour (tan), two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test substance did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 143 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin and required no classification.