Zinc di(benzimidazol-2-yl) disulphide

Endpoint summary

Description of key information

Based on the results of an OECD 439 study and an OECD 492 study the test item is not requiring classification for eye or skin irritation (UN GHS: No Category) (TOXI-COOP, 2017; LAUS 2017).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

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)

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.

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

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

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

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

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.

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:

2017-09-11 to 2017-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Species:

other: reconstructed human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 27001
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 hours at 37 °C

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.

Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, the controls and a defined amount of the test item were applied in duplicate in 1-min- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

- RhCE tissue construct used, including batch number

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27001
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 mg (test substance), 50 µL (controls)
- Duration and temperature of exposure; post-exposure incubation periods: 6 hours at 37 ± 1 °C; 25 min at room temperature + 18 hours at 37 ± 1 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary. Furthermore, no colour development was visible. Therefore, the main test was performed without colourant controls.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a Microtiter plate photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is: < 50 % of negative control
3. Acceptable variability between tissue replicates: < 20 %

Irritation parameter:

other: % Viability

Run / experiment:

Tissue 1

Value:

99.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % Viability

Run / experiment:

Tissues 2

Value:

105.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD = 1.6
- Acceptance criteria met for positive control: Yes, % mean relative viability of positive control was 42.2 %
- Variation within replicates:
0.2% (negative control)
3.5% (positive control)
6.0% (test item)

Table 1 Results

Group	Tissue 1		Tissue 2		Mean
	Viability %	OD	Viability %	OD	Viability %
Negative control	100	1.620	100	1.624	100
Positive control	44	0.713	40.5	0.657	42.2
Test substance	99.1	1.607	105.1	1.705	102.1

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was increased to 102.1 %. This value is above the threshold for eye irritation potential (≤ 60%), i.e. according to OECD 492 is identified as not irritating.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD guideline 492 (LAUS, 2017).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours. 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 25 minutes and further 18 hours with fresh medium.

After treatment with the negative control (sterile deionized water) the mean OD was 1.6 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 42.2 % (study acceptance criterion: <50%).Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 102.1 % and, thus, lower above 60%, i.e. according to OECD 492 the test item is identified as non irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

in vitro skin irritation

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 (TOXICOOP, 2017).

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.

in vitro eye irritation

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD guideline 492 (LAUS, 2017).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours. 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 25 minutes and further 18 hours with fresh medium.

After treatment with the negative control (sterile deionized water) the mean OD was 1.6 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 42.2 % (study acceptance criterion: <50%).Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 102.1 % and, thus, above 60%, i.e. according to OECD 492 the test item is identified as non irritant to the eye.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for eye or skin irritation under Regulation (EC) No 1272/2008.