Zinc di(benzimidazol-2-yl) disulphide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-30 to 2017-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

28 July 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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc di(benzimidazol-2-yl) disulphide

EC Number:

221-203-2

EC Name:

Zinc di(benzimidazol-2-yl) disulphide

Cas Number:

3030-80-6

Molecular formula:

C7H6N2S.1/2Zn

IUPAC Name:

2-{[(1H-1,3-benzodiazol-2-ylsulfanyl)zincio]sulfanyl}-1H-1,3-benzodiazole

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No. of test material: OUCHI SHINKO CHEMICAL INDUSTRIAL CO., LTD.; 608011
- Expiration date of the lot/batch: 2018-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep to the storeroom with suitable ventilation. Avoid fire, direct sunlight and moisture. Store at room temperature.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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 (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin (TM) SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model
- Tissue batch number(s): 17-EKIN-035
- Expiry date: 04 September 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume washing steps: 25 mL PBS 1 x solution
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

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 corrosive to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

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

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq. solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

77

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

73

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

83

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean OD = 1.035)
- Acceptance criteria met for positive control: Yes (mean OD = 0.262)
- Acceptance criteria met for variability between replicate measurements: Yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance did not show any skin irritating properties under the utilized testing conditions.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item Zinc di(benzimidazol-2-yl) disulphide on reconstituted human epidermis in the EPISKIN model in vitro according to OECD guideline 439 (TOXI-COOP, 2018).

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 crystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

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 chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item Zinc di(benzimidazol-2-yl) disulphide did not show significantly reduced cell viability in comparison to the negative control (mean viability: 78 %). 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.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.