(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-06 - 2018-03-09 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

OECD Guidelines for Testing of Chemicals Method 442D (In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method; adopted February 2015)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
In vitro methods have to be considered under REACH prior to conducting in vivo methods.

Test material

Test material information

Constituent 1

Reference substance name:

(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate

EC Number:

257-581-0

EC Name:

(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate

Cas Number:

51988-14-8

Molecular formula:

C9H6N2OS3

IUPAC Name:

phenyl[(5-sulfanylidene-4,5-dihydro-1,3,4-thiadiazol-2-yl)sulfanyl]methanone

Test material form:

solid: particulate/powder

Remarks:

white powder

Details on test material:

Storage Conditions : 15 to 30°C, protected from light

In vitro test system

Details on the study design:

TEST SYSTEM
Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D (batch number 55, passage 11 for Experiment 1, batch number 57, passage 12 for Experiment 2 and batch number 59, passage 11 for Experiment 3).
Identification:
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative/untreated control.
Preparation of Cultures:
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 9% foetal bovine serum and 1% Geneticin.

Results and discussion

Positive control results:

valid; see tables below

In vitro / in chemico

Results

Results 1

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

3.24

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: overall dose response for luciferase induction

Results 2

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

2.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: overall dose response for luciferase induction

Results 3

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

1.22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no unit

Results 4

Key result

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no unit

Results 5

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

412.26 µM

Cell viability:

84.92%

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: overall dose response for luciferase induction

Results 6

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

311.58 µM

Cell viability:

59.45%

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: overall dose response for luciferase induction

Results 7

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Cell viability:

not applicable

Remarks on result:

no relevant increase

Results 8

Key result

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

1 000 µM

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 9

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

1 445.82 µM

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 10

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

668.23 µM

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 11

Run / experiment:

run/experiment 3

Parameter:

IC50 [442D]

Value:

1 966.93 µM

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

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

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The study was performed according to OECD TG 442D under GLP on the registered substance itself. Positive and negative control were valid. The study was conducted to investigate the potential of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. In case of a positive response however, it may serve as standalone test. All four conditions for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 µM, dose response) were met in Experiment 1, however only three of the conditions were met in Experiment 2. Cell viability in Experiment 2 at the lowest concentration with induction of luciferase activity above 1.5-fold did not exceed 70% as specified in the protocol. However, all other results indicate that the test article is predicted as positive in this assay.
The test article, (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate, was thus considered to be positive in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted according to OECD 442D under GLP to investigate the potential of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in dimethylformamide (DMF) to the final stock concentration (200 mM). Serial dilutions were then made using DMF to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM.

Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the luciferase plates were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read.

 

The results are summarised as follows:

Criteria	Experiment 1	Experiment 2	Experiment 3
Imax	3.24	2.42	1.22
Cell Viability	84.92%	59.45%	Not calculated
EC1.5	412.26	311.58	Not calculated
Dose Response	Yes	Yes

 

All assay acceptance criteria were met, with the exception that the average induction for the positive control at 64 μM in Experiment 1 was 13.40. The assay was considered valid as there was a clear dose response with the positive control.

The Imax in Experiment 3 was less than 1.5 therefore this experiment gave a negative prediction.

All four conditions for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 µM, dose response) were met in Experiment 1, however, only three of the conditions were met in Experiment 2. Cell viability in Experiment 2 at the lowest concentration with induction of luciferase activity above 1.5-fold did not exceed 70% as specified in the protocol. However, all other results indicate that the test article is predicted as positive in this assay.

The test article,(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate, was thus considered to be positive in the ARE-Nrf2 Luciferase Test.