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

Endpoint summary

Description of key information

Skin sensitisation OECD 422: OECD 422D, GLP compliance, cell viability: 70%

Skin senitisation QSAR: in silico protein binding, positive

Skin sensitisation LLNA: OECD 429, study technically not feasible due to the instability of the test item in suitable vehicles.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Remarks:

in silico prediction of protein-binding

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

18 FEB 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox v4.5

2. MODEL (incl. version number)
Protein binding alerts by OASIS v 3.8
Protein binding alerts by OECD v 2.3
Protein binding potency Cys (DPRA 13%) v1.0
Protein binding potency Lys (DPRA 13%) v1.0
Protein binding alerts for skin sensitization according to GHS v 1.2
Protein binding alerts for skin sensitization by OASIS v 2.7
Protein Binding Potency h-CLAT v1.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
O=C(SC1SC(=S)NN=1)c1ccccc1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The skin sensitisation AOP is at least qualitatively well characterized, as the seminal events are generally accepted by the scientific community. The molecular initiating event (protein binding reactions) is based on long-standing, well-studied organic chemical mechanisms and reactions. Sensitisation is causally linked to keratinocyte activity and T-cell proliferation and, to a lesser extent, dendritic cell activation/maturation.
Computational or in silico techniques to predict chemical reactivity have been developed; they vary in complexity from the relatively simple approach of forming chemical categories from 2D structural alerts (i.e. SARs for qualitative identification of chemical sub-structures with the potential of being reactive), such as used in the OECD QSAR Toolbox to QSAR models (i.e. quantitative prediction of relative reactivity). For more model-specific information, please refer to field 'Principles of method if other than guideline'.

5. APPLICABILITY DOMAIN
Protein binding by OASIS v 3.8
The scope of the profiler is to investigate presence of alerts within target molecules responsible for interaction with proteins. The list of 112 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Protein binding by OECD v 2.3
The protein binding by OECD profiler contains 16 mechanistic alerts covering 52 structural alerts. These data are supported by mechanistic chemistry and references to the scientific literature (the meta data).

Protein binding potency Cys (DPRA 13%) v1.0
The profile contains 77 structural alerts extracted from about 229 chemicals with experimentally measured cysteine depletion values. The set of 77 structural alerts are separated into three potency categories: DPRA above 21% (DPRA 13%), DPRA less than 9% (DPRA 13%) and Grey zone 9-21% (DPRA 13%). Classification of potency categories is based on analysis published in a collaboration with L`Oreal (Dimitrov et al., 2016).

Protein binding potency Lys (DPRA 13%) v1.0
The profile contains 73 structural alerts extracted from about 228 chemicals with experimentally measured lysine depletion values. The set of 73 structural alerts are separated into three potency categories: DPRA above 21% (DPRA 13%), DPRA less than 9% (DPRA 13%) and Grey zone 9-21% (DPRA 13%).

Protein binding alerts for skin sensitization according to GHS v 1.2
The protein binding alerts are extracted from 517 chemicals used as a training set for the model. In the current version of the profiler are available 131 alerts, classifying chemicals into GHS categories 1A and 1B. Each alert is associated with reaction mechanistic domain, e.g. Schiff base formation, Michael addition, Acylation, etc. and it is specified in the help file supporting the mechanism of interaction of the alerting group with skin proteins.

Protein binding alerts for skin sensitization by OASIS v 2.7
This profiler accounts for incapability of some chemicals having an alert to interact with skin due to electronic and steric factors. This is explicitly defined by inhibition masks associated with some alerts. The list of 110 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Protein Binding Potency h-CLAT v1.0
The profile contains 30 structural alerts extracted from 223 chemicals with positive/negative data values. In cases of chemicals with available more than one value, the positive value has been used in the boundaries development.
The profiling results outcome assigns a target to the corresponding potency category based on matched structural criteria.

Guideline:

other: ECHA guidance R.6

Version / remarks:

May 2008

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR Toolbox v4.5
- Model(s) used: Protein binding by OASIS, Protein binding by OECD, Protein binding alerts for skin sensitization by OASIS, Protein Binding Potency h-CLAT, Protein binding potency Cys (DPRA 13%), Protein binding potency Lys (DPRA 13%), Protein binding alerts for skin sensitization according to GHS
- Model description:

Protein binding by OASIS: The scope of the profiler is to investigate presence of alerts within target molecules responsible for interaction with proteins. The list of 112 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Protein binding by OECD: The Protein binding by OECD profiler was developed by an analysis of direct acting structural alerts based on theoretical organic chemistry (the profiler does not contain metabolically / abiotically activated structural alerts). The alert compilations were analysed in order to place the information contained within the literature into a mechanistic chemistry framework. This mechanistic chemistry can be used as the basis for chemical category formation when utilising the Protein binding by OECD profiler. Within each of the five mechanistic domains related structural alerts have been grouped based on the presence of a common reactivity site into so-called mechanistic alerts. Chemical category formation can be carried out at either the mechanistic alert or structural alert level using this profiler.

Protein binding alerts for skin sensitization by OASIS: The scope of this profiler is to investigate the presence of alerts within the target molecules responsible for interaction with proteins and especially with skin proteins. This profiler accounts for incapability of some chemicals having an alert to interact with skin due to electronic and steric factors. This is explicitly defined by inhibition masks associated with some alerts. The list of 110 structural alerts has been separated into 11 mechanistic domains. Each of the mechanistic domains has been separated into more than 2 mechanistic alerts. The profiling result outcome assigns a target to the corresponding structural alert, mechanistic alerts and domain.

Protein binding potency Cys (DPRA 13%)
This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from Direct Peptide Reactivity Assay (DPRA). The DPRA is a reactivity assay which evaluates the ability of chemicals to react with proteins. As model peptides are used reduced glutathione and two synthetic peptides – lysine and cysteine. The reaction time for both lysine and cysteine is 24 hours. The peptide reactivity is reported as percent peptide depletion.

Protein binding potency Lys (DPRA 13%)
This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from Direct Peptide Reactivity Assay (DPRA). The DPRA is a reactivity assay which evaluates the ability of chemicals to react with proteins. As model peptides are used reduced glutathione and two synthetic peptides – lysine and cysteine. The reaction time for both lysine and cysteine is 24 hours. The peptide reactivity is reported as percent peptide depletion.

Protein binding alerts for skin sensitization according to GHS: The profiler is based on recently developed OASIS TIMES model for predicting skin sensitization according to GHS criteria. The profiler consists of structural and parametric (log KOW) requirements.

Protein Binding Potency h-CLAT
This profile is built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization. It is developed on the base of data derived from the human cell line activation (h-CLAT) assay. The h-CLAT is an in vitro method proposed to address the third key event (dendritic cell activation) of the skins sensitisation AOP by quantifying changes in the expression of cell surface markers associated with the process of activation of DC (i.e. CD86 and CD54), in the human leukemia cell line THP-1, following exposure to sensitizers. The measured expression levels of CD86 and CD54 cell surface markers are then used for supporting the discrimination between skin sensitisers and non-sensitisers.

- Justification of QSAR prediction: see field 'Justification for type of information'

Specific details on test material used for the study:

Sc1nnc(SC(=O)c2ccccc2)s1

Details on the study design:

Specific profilers were applied to assess the potential of protein-binding, a crucial mode of action to induce skin sensitisation.

Group:

test chemical

Remarks on result:

positive indication of skin sensitisation

Remarks:

Activated alkyl ester and thioester could interact with proteins via nucleophilic substitution on activated carbon atom. This predicted mode of action provides positive indication for skin sensitisation.

Table 1: Data matrix of profiling for protein binding with OECD Toolbox v4.5

		Chemical #1
Substance identity
CAS number		51988-14-8
Chemical name		(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate
Other identifier
SMILES		O=C(SC1SC(=S)NN=1)c1ccccc1
Profilers
General Mechanistic
Protein binding by OECD		Acylation Acylation >> Direct Acylation Involving a Leaving group Acylation >> Direct Acylation Involving a Leaving group >> Acetates
Protein binding potency Cys (DPRA 13%)		Out of mechanistic domain
Protein binding by OASIS		Acylation Acylation >> Direct acylation involving a leaving group Acylation >> Direct acylation involving a leaving group >> (Thio)Acetates Acylation >> Direct acylation involving a leaving group >> Azlactones and unsaturated lactone derivatives
Protein binding potency Lys (DPRA 13%)		Out of mechanistic domain
Endpoint Specific
Protein Binding Potency h-CLAT		No alert found
Protein binding alerts for skin sensitization by OASIS		Acylation Acylation >> Direct acylation involving a leaving group Acylation >> Direct acylation involving a leaving group >> (Thio)Acetates Acylation >> Direct acylation involving a leaving group >> Azlactones and unsaturated lactone derivatives
Protein binding alerts for skin sensitization according to GHS		Skin sensitization Category 1A Skin sensitization Category 1A >> Activated (thio)esters

Conclusions:

Based on the chemical structure, activated alkyl ester and thioester could interact with proteins via nucleophilic substitution on activated carbon atom. This predicted mode of action provides positive indication for skin sensitisation.

Executive summary:

Endpoint specific profilers was applied to assess the potential of protein-binding, a crucial mode of action to induce skin sensitisation. The applied profilers are designed to indicate chemicals could interact with proteins and could cause skin sensitization. The structural alerts in the endpoint specific profilers are focussed on chemistry associated with covalent binding to skin proteins associated with skin allergy.

Based on the chemical structure, activated alkyl ester and thioester could interact with proteins via nucleophilic substitution on activated carbon atom. This predicted mode of action provides positive indication for skin sensitisation.

 

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

Pre-test on feasibility of the conduction of a local lymph node assay

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-05-24 - 2019-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

pre-test

Deviations:

not applicable

Principles of method if other than guideline:

Feasibility testing by validation of the method for chemical analysis of the formulations in DMF

GLP compliance:

not specified

Type of study:

other: pre-test for modified LLNA

Justification for non-LLNA method:

The present report describes the pre-test for a modified LLNA, which cannot be conducted because the solvent is not suitable

Species:

other: none

Strain:

other: not applicable

Details on test animals and environmental conditions:

not applicable

Interpretation of results:

study cannot be used for classification

Conclusions:

The performance of the local lymph node assay was technically not feasible due to the instability of the test item in suitable vehicles. This was proven during validation of the method for chemical analysis of the formulations in DMF. The study was terminated in agreement with the Sponsor. In the course of in chemico/in vitro skin sensitisation testing it was shown, that neither water, acetone nor DMSO were suitable as vehicles due to insolubility of the test item.

Executive summary:

In the pre-test for the LLNA it was shown that the performance of the local lymph node assay was technically not feasible due to the instability of the test item in suitable vehicles. This was proven during validation of the method for chemical analysis of the formulations in DMF. In the course of in chemico/in vitro skin sensitisation testing it was shown, that neither water, acetone nor DMSO were suitable as vehicles due to insolubility of the test item.

Reference 3

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

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.

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.

Positive control results:

valid; see tables below

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

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

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

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

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

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

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Cell viability:

not applicable

Remarks on result:

no relevant increase

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation OECD 422D

The study was conducted according to OECD 442D under GLP to investigate the potential of test item to induce genes that are regulated by the antioxidant response element (ARE). The test article was dissolved in dimethylformamide (DMF) to the final stock concentration (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 and incubated at 37±1°C, 5% (v/v) CO2in air, in a humidified environment for 48±1 hours. 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 was thus considered to be positive in the ARE-Nrf2 Luciferase Test.

Skin sensitisation - in silico prediction of skin sensitisation

Protein binding of the registered substance was predicted with the relevant profilers implemented in the OECD QSAR Toolbox v4.5. Protein binding is the first key event to elicit skin sensitisation. Various profilers revealed positive indication for protein binding of the registered substance. This provides high evidence that the substance is positive for protein binding. 

Skin sensitisation LLNA

In the pre-test for the LLNA it was shown that the performance of the local lymph node assay was technically not feasible due to the instability of the test item in suitable vehicles. This was proven during validation of the method for chemical analysis of the formulations in DMF. In the course of in chemico/in vitro skin sensitisation testing it was shown, that neither water, acetone nor DMSO were suitable as vehicles due to insolubility of the test item.

Conclusion

Within the frame of a '2 out of 3 approach' addressing two key events of skin sensitsation (in vitro keratinocyte gene expression and in silico protein binding) having congruent results (positive) the substance is concluded to be skin sensitising.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available data on skin sensitisation, the registered substance is classified as 'Skin Sens Cat. 1' under Regulation (EC) No 1272/2008. The available data does not allow for sub-categorisation.