Tetrahydrothiophene

Endpoint summary

Description of key information

The skin sensitization potential of tetrahydrothiophene was evaluated in a battery of 3 in vitro assays, using QSAR models and by read across to analogue substances.

In vitro assays

The 3 in vitro tests are part of a tiered strategy for skin sensitization assessment.

The ability of tetrahydrothiophene to induce an increase in cell surface markers expression in THP-1 cells was evaluated using the h-CLAT test method (Gerbeix, 2017). The design of this study was based on the guideline OECD Guideline 442E and the study was performed in compliance with the OECD Principles of Good Laboratory Practice. A solubility assessmentwas first performed in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations. Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay in order to select sub-toxic concentrations for testing in the main test. The skin sensitizing potential of the test item was then tested in the main test in four successive runs (three validated and one non-validated runs. In each run, the test item formulations were applied to triplicate wells containing THP-1 cells and cultured for 24h ± 30 minutes at 37°C, 5% CO₂in a humidified incubator. A set of control wells (each in triplicate) was also added in each plate to guarantee the validity of each run. At the end of the incubation period, all cells from the first wells were labeled with IgG1-FITC antibodies, the second ones were labeled with CD86-FITC antibodies and the third ones were labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination. For each run, the Mean Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

Tetrahydrothiophene was found soluble in DMSO at the concentration of 500 mg/mL. It did not decrease cell viability < 75% during both DRF runs. No mean CV75 value was therefore calculated, and the highest tested concentration retained for use in the main test was 1000 µg/mL.Based on the prediction model presented in the OECD 442E guideline, and as positive outcomes were noted on both markers during run A and one of them during run D, combined results from all validated runs resulted in a global positive outcome.

 

Tetrahydrothiophene

RFI for CD86

RFI for CD54

Viability (%)

Run conclusion

Conc.(µg/mL)

A

B

C

D

A

B

C

D

A

B

C

D

A

B

C

D

279.08 334.90 401.88 482.25 578.70 694.44 833.33 1000.00

125 107 147 165 145 161 131 112

103 71 73 100 143 129 149 81

100 64 102 101 94 97 113 76

104 89 97 128 120 131 128 89

94 94 212 135 218 188 182 -57

76 82 91 118 106 103 124 85

106 -25 100 113 113 181 188 113

127 138 146 196 204 231 227 131

93.1 94.5 91.7 90.6 88.3 82.6 78.9 91.1

94.7 96.6 96.6 91.9 89.4 87.4 82.8 95.1

94.0 96.9 91.8 89.1 88.0 85.3 84.1 93.1

96.4 97.5 96.9 92.4 90.4 88.5 86.0 94.7

P1,2

N

I

P2

Positive

 

N = run with negative outcome	P1 = run with positive outcome in CD86	Grey font (run C): invalidated run due to DNCB positive control not reaching acceptance criteria
Inc = Inconclusive run	P2 = run with positive outcome in CD54
I = Invalidated run	P1,2 = run with positive outcome in CD86 and CD54

 

Tetrahydrothiophene was found to be POSITIVE in the h-CLAT.

 

The potential of the tetrahydrothiophene to activate the Nrf2 transcription factor was evaluated using the KeratinoSens test method (Chevallier, 2017). The study was performed according to the OECD guideline No. 442D and in compliance with the principles of Good Laboratory Practice. This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent validated runs were performed as part of this study. For each run, the test item was diluted in DMSO at 200 mM. All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated. Both validated runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO. 

At these tested concentrations:

.              no precipitate/emulsion of the test item was observed in any wells at the end of the 48-hour treatment period in either run,

.              no noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70% in both runs), therefore no geometric mean IC30 or IC50 was calculated,

.              no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run. Moreover, the Imax values were < 1.5 in both runs (i.e.0.95 and 1.49 in the first and second runs, respectively).

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential. Under the experimental conditions of this study, tetrahydrothiophene was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

 

The reactivity of tetrahydrothiophene to synthetic cysteine and lysine peptides was evaluated the DPRA test method (Chevallier, 2017). The study was performed according to the OECD guideline No. 442C and in compliance with the principles of Good Laboratory Practice. The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent). The test item was diluted at 100 mM in acetonitrile. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item co-eluted with the cysteine peptide (% interference of 195.2%). Since the corresponding peak cannot be integrated and since the determination of reactivity could not be based on the percent depletion data from the lysine reaction alone, the peptide reactivity analysis was considered as inconclusive and the test item as incompatible with the DPRA test. Under the experimental conditions of this study, since the tetrahydrothiophene co-eluted with the cysteine peptide, it was considered as incompatible with the DPRA test and therefore its peptide reactivity analysis was inconclusive.

No definitive conclusion can be drawn from this in vitro test battery, considering the positive result in the h-CLAT assay, the negative result in the KeratinoSens assay and the incompatibility with the DPRA assay.

QSAR assessment

The allergic contact dermatitis potential of tetrahydrothiophene in guinea pig and human was evaluated via the Danish QSAR database using MultiCASE Ultra, Leadscope Enterprise and SciMatics SciQSAR versions of commercial CASE Ultra model A33. No indication of skin sensitisation was reported, tetrahydrothiophene being inside the applicability domain of each model.

 

Read across

The presence of structural alert of skin sensitisation in the molecular structure of tetrahydrothiophene (THT), Tetrahydrothiophene-1,1-dioxide (skin metabolite), Thiophene (structural analogue), Dimethyl sulfide, Ethyl methyl sulphide, Diethyl sulphide and 2,2-bis(methylthio)propane (diakyl sulfide family) was evaluated with 2 QSAR models. The Toxtree (Version 2.6.13) captures electrophilic mechanistic information and include “Michael-type addition reaction”, “Schiff base formation”, “acylation”, “nucleophilic aromatic substitution” (SNAr) and “second order nucleophilic aliphatic substitution” (SN2). The OECD QSAR Toolbox (Version 4.0) capture electrophilic mechanistic information and include " Keratinocyte gene expression", "Protein binding alerts for skin sensitization by OASIS v1.4", "Protein Binding Potency h-CLAT", "Protein binding by OASIS v1.4", Protein binding by OECD", "Protein binding potency", "Protein binding potency Cys (DPRA 13%)" and "Protein binding potency Lys (DPRA 13%)". No skin sensitisation reactivity domains alerts were identified for any of these compouunds.

Tetrahydrothiophene-1,1-dioxide, Thiophene, Dimethyl sulfide, Ethyl methyl sulphide, Diethyl sulphide and 2,2-bis(methylthio)propane were tested in vivo for skin sensitisation. No skin sensitisation potential was identified for any of these products.

 

Conclusion

No definitive conclusion can be drawn from the battery of in vitro assays performed with tetrahydrothiophene. No allergic contact dermatitis potential in guinea pig and human was identified for tetrahydrothiophene by the Danish QSAR database. No reactivity domains alerts were identified by QSAR models for Tetrahydrothiophene, its skin metabolites and structural analogues, which were all negative in in vivo skin sensitisation assays. Therefore, it is not expected that tetrahydrothiophene could be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 March 2017 - 24 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Test systems:

1/ Cystein peptide:
Peptide sequence: Ac-RFAACAA-COOH
Peptide sequence synonyms: Ac-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
Molecular weight: 750.88 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 111016HS_MHeW0117
Storage condition: at -20°C
Description : white powder

2/ Lysine peptide
Peptide sequence: Ac-RFAAKAA-COOH
Peptide sequence synonyms: Ac-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
Molecular weight: 775.91 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 220114HSDWW0117
Storage condition: at -20°C
Description: white powder


Vehicle used: acetonitrile
Positive control: cinnamaldehyde
Criteria: Interpretation of results

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

Run / experiment:

other: Single run

Parameter:

other: Mean of cysteine and lysine % depletion

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Inconclusive outcome due to coelution of test item with cysteine peptide.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r2) => 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
- for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.

The test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Interpretation of results:

study cannot be used for classification

Conclusions:

Since the test item co-eluted with the cysteine peptide, it was considered as incompatible with the DPRA test and therefore its peptide reactivity analysis was inconclusive.

Executive summary:

The objective of this study was to evaluate the reactivity of tetrahydrothiophene to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.  The study was performed according to the international OECD guideline No. 442C and in compliance with the principles of Good Laboratory Practice. The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent). The test item was diluted at 100 mM in acetonitrile.The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item co-eluted with the cysteine peptide (% interference of 195.2%). Since the corresponding peak cannot be integrated and since the determination of reactivity could not be based on the percent depletion data from the lysine reaction alone, the peptide reactivity analysis was considered as inconclusive and the test item as incompatible with the DPRA test. Under the experimental conditions of this study, since the tetrahydrothiophene co-eluted with the cysteine peptide, it was considered as incompatible with the DPRA test and therefore its peptide reactivity analysis was inconclusive.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 March 2017 - 30 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: activation of the Nrf2 transcription factor of the keratinocytes

Details on the study design:

In vitro test system:
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid.
Supplier: this cell line was provided by Givaudan.

Control items:
Vehicle and negative control: DMSO
Positive control: Cinnamic aldehyde (CA).

On the basis of solubility results, the test item was diluted in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 to obtain a total of 12 concentrations in a 96-well plate; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.

Study design:
Solubility assay
The test item was found soluble in DMSO at 200 mM.

Treatment
The test item was tested in two independent runs using cells from a different passage number.
The plates were incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

End-point measurements
- Microscopic observation to evaluate the presence or absence of precipitate,
- Luminescence flash signal to evaluate induction signal,
- Absorbance signal to evaluate the cytotoxicity.

RESULTS ANALYSIS
Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing was performed automatically.

For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted.
For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.

For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
- Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
- EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
- IC50 and IC30: concentrations effecting a reduction of cellular viability by 50% and 30%,
- Indication whether significant 1.5-fold gene induction occurred below the IC30.

The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose response curves or large variation may lead to wrong extrapolations.

Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

Evaluation criteria of the test item
The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Run / experiment:

mean

Parameter:

other: EC1.5

Remarks:

No data available since Imax < 1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated.
In the second run, the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax of 16.05). However, since a clear dose response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.

Both validated runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:
- no precipitate/emulsion of the test item was observed in any wells at the end of the 48-hour treatment period in either run,
- no noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70% in both runs), therefore no geometric mean IC30 or IC50 was calculated,
- no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run. Moreover, the Imax values were < 1.5 in both runs (i.e. 0.95 and 1.49 in the first and second runs, respectively).

Interpretation of results:

other: negative

Conclusions:

Tetrahydrothiophene was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The potential of the tetrahydrothiopene to activate the Nrf2 transcription factor was evaluated in a study performed according to the OECD guideline No. 442D and in compliance with the principles of Good Laboratory Practice. This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent validated runs were performed as part of this study. For each run, the test itemwas diluted in DMSO at 200 mM. All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated. Both validated runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO. 

At these tested concentrations:

.            no precipitate/emulsion of the test item was observed in any wells at the end of the 48-hour treatment period in either run,

.            no noteworthy decrease in cell viability was noted in either run (i.e. cell viability > 70% in both runs), therefore no geometric mean IC₃₀or IC₅₀ was calculated,

.            no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in either run. Moreover, the I_maxvalues were < 1.5 in both runs (i.e. 0.95 and 1.49 in the first and second runs, respectively).

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential. Under the experimental conditions of this study, tetrahydrothiophene was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 March 2017 - 16 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation - Human Cell Line Activation Test (h-CLAT)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Test system: THP-1 cell line (ref TIB-202) from ATCC (Manassas, USA)
Vehicle control: DMSO
Positive controls: 2,4-Dinitrochlorobenzene (DNCB) and Nickel Sulfate (NiSO4)

Study Design:
Solubility assay in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used.
Then, Dose-Range Finding assay in order to select sub-toxic concentrations for testing in the main test.
Then, at least 2 runs of main test for measurement of Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is = 150 at any concentration leading to = 50% viability,
- RFI of CD54 is = 200 at any concentration leading to = 50% viability.

In other circumstances, the run is considered as negative.

Run / experiment:

other: Run A

Parameter:

other: Max RFI CD86

Value:

165

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Run positive for CD86

Run / experiment:

other: Run A

Parameter:

other: Max RFI CD54

Value:

218

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Run positive for CD54

Run / experiment:

other: Run B

Parameter:

other: Max RFI CD86

Value:

149

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Run negative for CD86

Run / experiment:

other: Run B

Parameter:

other: Max RFI CD54

Value:

124

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Run negative for CD54

Run / experiment:

other: Run C

Parameter:

other: Max RFI CD86

Value:

113

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other:

Remarks:

Run invalidated due to DNCB positive control not reaching acceptance criteria.

Run / experiment:

other: Run C

Parameter:

other: Max RFI CD54

Value:

188

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other:

Remarks:

Run invalidated due to DNCB positive control not reaching acceptance criteria.

Run / experiment:

other: Run D

Parameter:

other: Max RFI CD86

Value:

131

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Run negative for CD86

Run / experiment:

other: Run D

Parameter:

other: Max RFI CD54

Value:

231

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Run positive for CD54

 

Tetrahydrothiophene

RFI for CD86

RFI for CD54

Viability (%)

Run conclusion

Conc.(µg/mL)

A

B

C

D

A

B

C

D

A

B

C

D

A

B

C

D

279.08 334.90 401.88 482.25 578.70 694.44 833.33 1000.00

125 107 147 165 145 161 131 112

103 71 73 100 143 129 149 81

100 64 102 101 94 97 113 76

104 89 97 128 120 131 128 89

94 94 212 135 218 188 182 -57

76 82 91 118 106 103 124 85

106 -25 100 113 113 181 188 113

127 138 146 196 204 231 227 131

93.1 94.5 91.7 90.6 88.3 82.6 78.9 91.1

94.7 96.6 96.6 91.9 89.4 87.4 82.8 95.1

94.0 96.9 91.8 89.1 88.0 85.3 84.1 93.1

96.4 97.5 96.9 92.4 90.4 88.5 86.0 94.7

P1,2

N

I

P2

Positive

 

N = run with negative outcome	P1 = run with positive outcome in CD86	Grey font (run C): invalidated run due to DNCB positive control not reaching acceptance criteria
Inc = Inconclusive run	P2 = run with positive outcome in CD54
I = Invalidated run	P1,2 = run with positive outcome in CD86 and CD54

 

Interpretation of results:

other: Positive

Conclusions:

The test item was found to be POSITIVE in the h-CLAT.

Executive summary:

The objective of the study was to determine the ability of tetrahydrothiophene to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.

The design of this study was based on the guideline OECD Guideline 442E and the study was performedin compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice. A solubility assessmentwas first performed in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations. Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay in order to select sub-toxic concentrations for testing in the main test. The skin sensitizing potential of the test item was then tested in the main test in four successive runs (three validated and one non-validated runs. In each run, the test item formulations were applied to triplicate wells containing THP-1 cells and cultured for 24h ± 30 minutes at 37°C, 5% CO₂ in a humidified incubator. A set of control wells (each in triplicate) was also added in each plate to guarantee the validity of each run. At the end of the incubation period, all cells from the first wells were labeled with IgG1-FITC antibodies, the second ones were labeled with CD86-FITC antibodies and the third ones were labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

Tetrahydrothiophene was found soluble in DMSO at the concentration of 500 mg/mL. It did not decrease cell viability < 75% during both DRF runs. No mean CV75 value was therefore calculated, and the highest tested concentration retained for use in the main test was 1000 µg/mL. Based on the prediction model presented in the OECD 442E guideline, and as positive outcomes were noted on both markers during run A and one of them during run D, combined results from all validated runs resulted in a global positive outcome.

 

Tetrahydrothiophene

RFI for CD86

RFI for CD54

Viability (%)

Run conclusion

Conc.(µg/mL)

A

B

C

D

A

B

C

D

A

B

C

D

A

B

C

D

279.08 334.90 401.88 482.25 578.70 694.44 833.33 1000.00

125 107 147 165 145 161 131 112

103 71 73 100 143 129 149 81

100 64 102 101 94 97 113 76

104 89 97 128 120 131 128 89

94 94 212 135 218 188 182 -57

76 82 91 118 106 103 124 85

106 -25 100 113 113 181 188 113

127 138 146 196 204 231 227 131

93.1 94.5 91.7 90.6 88.3 82.6 78.9 91.1

94.7 96.6 96.6 91.9 89.4 87.4 82.8 95.1

94.0 96.9 91.8 89.1 88.0 85.3 84.1 93.1

96.4 97.5 96.9 92.4 90.4 88.5 86.0 94.7

P1,2

N

I

P2

Positive

 

N = run with negative outcome	P1 = run with positive outcome in CD86	Grey font (run C): invalidated run due to DNCB positive control not reaching acceptance criteria
Inc = Inconclusive run	P2 = run with positive outcome in CD54
I = Invalidated run	P1,2 = run with positive outcome in CD86 and CD54

 

Tetrahydrothiophene was found to be POSITIVE in the h-CLAT.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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

Guideline:

other: Chapter R.6: QSARs and grouping of chemicals

Principles of method if other than guideline:

MultiCASE CASE Ultra commercial model A33 for allergic contact dermatitis (ACD) in guinea pig and human (MultiCASE CASE Ultra 1.4.6.6 64-bit)
Leadscope Enterprise version of commercial CASE Ultra model A33 for allergic contact dermatitis (ACD) in guinea pig and human (Leadscope Predictive Data Miner, a component of Leadscope Enterprise version 3.1.1-10.)
SciMatics SciQSAR version of commercial CASE Ultra model A33 for allergic contact dermatitis (ACD) in guinea pig and human (SciQSAR version 3.1.00.)

Specific details on test material used for the study:

Smiles: C1CCSC1

Parameter:

other: Leadscope

Remarks on result:

no indication of skin sensitisation

Remarks:

inside applicability domain

Parameter:

other: SciQSAR

Remarks on result:

no indication of skin sensitisation

Remarks:

inside applicability domain

Parameter:

other: CASE Ultra

Remarks on result:

no indication of skin sensitisation

Remarks:

inside applicability domain

Interpretation of results:

other: negative

Executive summary:

The allergic contact dermatitis potential of tetrahydrothiophene in guinea pig and human was evaluated via the Danish QSAR database using MultiCASE Ultra, Leadscope Enterprise and SciMatics SciQSAR versions of commercial CASE Ultra model A33. No indication of skin sensitisation was reported, tetrahydrothiophene being inside the applicability domain of each model.

Reference 5

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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
Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach)

2. MODEL (incl. version number)
Version 2.6.13

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES: C1CCSC1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Skinsensitisation
- Unambiguous algorithm: see attached rules
- Defined domain of applicability: rules were developed for the identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach
- Appropriate measures of goodness-of-fit and robustness and predictivity: not relevant
- Mechanistic interpretation: according to the rules and decision tree of the specific end-point

5. APPLICABILITY DOMAIN
- Descriptor domain: Defined by the rules
- Structural and mechanistic domains: Defined by the rules
- Similarity with analogues in the training set: not relevant

6. ADEQUACY OF THE RESULT
The estimation is coherent with the available in vivo data

Qualifier:

according to guideline

Guideline:

other: REACH guidance. Chapter R.6: QSARs and grouping of chemicals

Version / remarks:

May 2008

Principles of method if other than guideline:

Toxtree (Version 2.6.13) alerts for skin sensitization capture electrophilic mechanistic information and include “Michael-type addition reaction”, “Schiff base formation”,“acylation”, “nucleophilic aromatic substitution” (SNAr) and “second order nucleophilic aliphatic substitution” (SN2). The final alert “Reactivity domain alert” is true if any other alert is true.

Specific details on test material used for the study:

Chemical name(s) CAS Reg no. Smiles
Tetrahydrothiophene (THT) 110-01-0 C1CCSC1
Tetrahydrothiophene-1,1-dioxide 126-33-0 O=S1(=O)CCCC1
Thiophene 110-02-1 c1ccsc1
Dimethyl sulfide 75-18-3 CSC
Ethyl methyl sulphide 624-89-5 CCSC
Diethyl sulphide 352-93-2 CCSCC
2,2-bis(methylthio)propane 6156-18-9 CSC(C)(C)SC

Details on the study design:

Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach.
Available since ToxTree 2.1.0 (under name "Skin sensitisation alerts" and "Skin sensitisation alerts (M.Cronin)"). The name is changed to "Skin sensitisation reactivity domain" by P&G team suggestion in order to reflect the fact the alerts provide grouping into reactivity mode of action and do not predict skin sensitisation potential.

Run / experiment:

other: All compounds

Parameter:

other: Toxtree

Remarks on result:

other: No skin sensitisation reactivity domains alerts identified

Run / experiment:

other: All compounds

Parameter:

other: QSNAR.SNAr-Nucleophilic Aromatic Substitution

Remarks on result:

other: No

Run / experiment:

other: All compounds

Parameter:

other: QSB.Schiff Base Formation

Remarks on result:

other: No

Run / experiment:

other: All compounds

Parameter:

other: QMA.Michael Acceptor

Remarks on result:

other: No

Run / experiment:

other: All compounds

Parameter:

other: QSN2.SN2-Nucleophilic Aliphatic Substitution

Remarks on result:

other: No

Other effects / acceptance of results:

 

Interpretation of results:

other: No structural alert for skin sensitisation

Executive summary:

The presence of structural alert of skin sensitisation in the molecular structure of tetrahydrothiophene (THT), Tetrahydrothiophene-1,1-dioxide (skin metabolite), Thiophene (structural analogue), Dimethyl sulfide, Ethyl methyl sulphide, Diethyl sulphide and 2,2-bis(methylthio)propane (diakyl sulfide family) was evaluated with Toxtree (Version 2.6.13). The alerts for skin sensitization capture electrophilic mechanistic information and include “Michael-type addition reaction”, “Schiff base formation”, “acylation”, “nucleophilic aromatic substitution” (SNAr) and “second order nucleophilic aliphatic substitution” (SN2). The final alert “Reactivity domain alert” is true if any other alert is true. No skin sensitisation reactivity domains alerts were identified for any of these compouunds.

Reference 6

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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:

QSAR Toolbox assesses the likelihood that the target substance will bind to protein using four structural feature recognition (“alert”) systems for protein binding.
- QSAR Toolbox recognizes structural features that have been identified as binding to skin proteins using four alert systems, termed “profilers.” Three of the profilers are general mechanistic profilers and one is an endpoint-specific (i.e., skin sensitization) mechanistic profiler.
. Protein binding by OASIS v1.4 identifies whether a substance contains any of 101 structural features (alerts) in 11 mechanistic domains that are responsible for the interaction with proteins.
. Protein binding by OECD determines whether a substance binds to proteins using a scheme that has five overarching mechanistic domains related to structural alerts grouped by the presence of a common reactivity site into mechanistic alerts. This profiler has 102 categories of protein binding that includes 16 mechanistic alerts (e.g., ring-opening acylation) that cover 52 structural alerts (e.g., alpha-lactams).
Protein binding potency assesses the likelihood that the target substance will covalently bind with the thiol group of glutathione. The profiler contains 49 Michael addition and 46 bimolecular nucleophilic substitution (SN2) categories. The 95 structural alerts are separated into five potency categories of reactivity: extremely, highly, moderately, slightly, and suspect.
. Protein binding alerts for skin sensitization by OASIS v1.4 is the endpoint-specific profiler that evaluates whether a substance binds to proteins. Compared to the protein binding by OASIS v1.4 profiler (discussed in the first bullet above), this profiler accounts for the inhibition of protein binding in the skin, produced by electronic and steric factors, for substances with protein binding alerts. It uses 100 structural alerts that have been categorized into 11 mechanistic domains. Each mechanistic domain has more than two mechanistic alerts.

The Toolbox also includes a specific profilers built in relation with the implementation of the adverse outcome pathway (AOP) for skin sensitization:
-Protein binding potency Cys or Lys (DPRA 13%): This profile was built in relation 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. 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). [S. Dimitrov, A. Detroyer, C. Piroird, C. Gomes, J. Eilstein, T. Pauloin, C. Kuseva, H. Ivanova, I. Popova, Y. karakolev, S. Ringeisses, O. Mekenyan, Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example. J Appl Toxicol, 2016, DOI 10.1002/jat.3318]
- h-CALT: This profile was 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. 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.
- Keratinocyte gene expression: This profile was built on the base of data derived from the KeratinoSens assay, which examined the potential for chemicals to induce the expression of a luciferase reporter gene under control of a single copy of the ARE element of the human AKR1C2 gene stably inserted into immortalized human keratinocytes. Relevance to skin sensitization is inferred from the relationship of Keap1-Nrf2-ARE regulatory pathway and its detection of electrophilic chemicals to sensitization. All included categories are defined by analysis in experimental data for EC1.5 values (the concentration eliciting a 1.5-fold increase in luciferase induction). No gene induction is observed when EC1.5 is >2000. The profile contains 18 structural alerts extracted from about 300 chemicals comprised within EC1.5 data. The set of 18 structural alerts are separated into four categories: very high gene expression, high gene expression, moderate gene expression and low gene expression. Classification of categories depends on the EC1.5 values and is as follows: chemicals having very high gene expression (EC1.5< = 15 uM); high gene expression (EC1.5 = 15 – 50 uM); moderate gene expression (EC1.5 = 50 – 100 uM); low gene expression (EC1.5 = 100 – 1999 uM). The profiling results outcome assigns a target to the corresponding potency category based on matched structural criteria.

If QSAR Toolbox identified no protein binding alerts for the target substance, a further assessment was conducted to determine if the substance might be a pre- or prohapten. The autoxidation and skin metabolism modules were used to generate oxidation/metabolism products. These products were then profiled as above for protein binding alerts.

If none were identified, the target substance was classified to be a non sensitizer

Qualifier:

according to guideline

Guideline:

other: Chapter R.6: QSARs and grouping of chemicals

Specific details on test material used for the study:

Chemical name(s) CAS Reg no. Smiles
Tetrahydrothiophene (THT) 110-01-0 C1CCSC1
Tetrahydrothiophene-1,1-dioxide 126-33-0 O=S1(=O)CCCC1
Thiophene 110-02-1 c1ccsc1
Dimethyl sulfide 75-18-3 CSC
Ethyl methyl sulphide 624-89-5 CCSC
Diethyl sulphide 352-93-2 CCSCC
2,2-bis(methylthio)propane 6156-18-9 CSC(C)(C)SC

Run / experiment:

other: All compounds

Parameter:

other: OECD QSAR Toolbox

Remarks on result:

other: No structural alert of skin sensitisation

Run / experiment:

other: All compounds

Parameter:

other: Keratinocyte gene expression

Remarks on result:

other: Not possible to classify according to these rules

Run / experiment:

other: All compounds

Parameter:

other: Protein binding alerts for skin sensitization by OASIS v1.4

Remarks on result:

other: No alert found

Run / experiment:

other: All compounds

Parameter:

other: Protein Binding Potency h-CLAT

Remarks on result:

other: No alert found

Run / experiment:

other: All compounds

Parameter:

other: Protein binding by OECD

Remarks on result:

other: No alert found

Run / experiment:

other: All compounds

Parameter:

other: Protein binding by OASIS v1.4

Remarks on result:

other: No alert found

Run / experiment:

other: All compounds

Parameter:

other: Protein binding potency

Remarks on result:

other: Not possible to classify according to these rules (GSH)

Run / experiment:

other: All compounds

Parameter:

other: Protein binding potency Cys (DPRA 13%)

Remarks on result:

other: DPRA less than 9% (DPRA 13%)|DPRA less than 9% (DPRA 13%) >> No protein binding alert

Run / experiment:

other: All compounds

Parameter:

other: Protein binding potency Lys (DPRA 13%)

Remarks on result:

other: DPRA less than 9% (DPRA 13%)|DPRA less than 9% (DPRA 13%) >> No protein binding alert

Interpretation of results:

other: No structural alert for skin sensitisation

Executive summary:

The presence of structural alert of skin sensitisation in the molecular structure of tetrahydrothiophene (THT), Tetrahydrothiophene-1,1-dioxide (skin metabolite), Thiophene (structural analogue), Dimethyl sulfide, Ethyl methyl sulphide, Diethyl sulphide and 2,2-bis(methylthio)propane (diakyl sulfide family) was evaluated with the OECD QSAR Toolbox (Version 4.0). The specific profiles for skin sensitization capture electrophilic mechanistic information and include " Keratinocyte gene expression", "Protein binding alerts for skin sensitization by OASIS v1.4", "Protein Binding Potency h-CLAT", "Protein binding by OASIS v1.4", Protein binding by OECD", "Protein binding potency", "Protein binding potency Cys (DPRA 13%)" and "Protein binding potency Lys (DPRA 13%)". No skin sensitisation reactivity domains alerts were identified for any of these compouunds.

Reference 7

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was performed the implementation of the REACH regulation

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-lès-Elbeuf, France
- Age at study initiation: ca 3-month old
- Weight at study initiation: 382 +/- 25 g for males and 361 +/- 16g for females
- Housing: polycarbonate cage
- Diet (e.g. ad libitum): 106 pelleted diet (UAR, 91360 Villemoisson-sur-orge, France)
- Water (e.g. ad libitum): filtered tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 30-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

5%/0.1 ml

Day(s)/duration:

Day 1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.5 ml

Day(s)/duration:

Day 8

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

50%/0.5 ml

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

5 males and 5 females in the control group
10 males and 10 females in the treated group

Details on study design:

Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).
On day 1, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed in the interscapular region.
On day 8, this same test site received a cutaneous application of the test substance (treated group) or the vehicle (control group) and was then covered by an occlusive dressing for 48 hours.
On day 22, after a rest period of 12 days, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours.
Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.
At the end of the study, animals were killed without examination of internal organs. No skin samples were taken from the challenge application sites.

The sensitivity of the guinea-pigs in CIT experimental conditions was checked with positive sensitizers DNCB and MERCAPTOBENZOTHIAZOLE. During the induction period, the test substance DNCB was applied at the concentrations of 0.1 % (w/w) (day 1) and 1 %(w/w) (day 8). The test substance MERCAPTOBENZOTHIAZOLE was applied at the concentrations of 1% (w/w) (day 1) and 20% (w/w) (day 8). For the challenge application, the test substance DNCB was applied at the concentration of 1 % (w/w). The test substance MERCAPTOBENZOTHIAZOLE was applied at the concentration of 20% (w/w).

Positive control substance(s):

yes

Remarks:

DNCB and MERCAPTOBENZOTHIAZOLE

Positive control results:

The species and strain which were used showed a satisfactory sensitization response in 90% animals treated with DNCB and in 30% animals treated with MERCAPTOBENZOTHIAZOLE.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Interpretation of results:

GHS criteria not met

Conclusions:

According to the maximization method of Magnusson and Kligman, THIOPHENE does not induce delayed contact hypersensitivity in guinea-pigs.

Executive summary:

Thiophene was tested in an OECD Test Guideline 406 skin sensitization study (maximization test) with guinea pigs. Induction was accomplished by an intradermal injection of a 5% solution of thiophene in Freund’s adjuvant on day 1 followed by a topical application of undiluted thiophene on day 8 (covered for 48 hours) to 10 animals/sex in the treated group and 5 animals/sex in the control. On day 22, a challenge dose of 50% solution of thiophene was applied topically (covered for 24 hours). Skin reactions were evaluated 24 and 48 hours following the removal of the dressing after the challenge dose. No clinical signs or deaths were observed. At 24 and 48 hours following the removal of the dressing after the challenge dose, no cutaneous reactions were recorded. It was concluded that thiophene is not a skin sensitizer. Satisfactory sensitization response was observed using the positive controls, dinitrochlorobenzene and mercaptobenzothiazole.

Reference 8

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Test performed before the implemenation of the REACH regulation

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: 1-2 months old
- Weight at study initiation:
Male: 352 +/- 12 g
Female: 354 +/-21 g
- Housing: housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm)
- Diet: free access to 106 pelleted diet (SAFE, Villemoisson, Epinay sur-Orge, France)
- Water: water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 to 70
- Air changes (per hr):12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 20 December 2006 to 02 February 2007

Route:

intradermal and epicutaneous

Vehicle:

other: corn oil (i.d. induction) and acetone (challenge)

Concentration / amount:

Induction:
- 10% in corn oil intradermal
- undiluted topically
Challenge:
10% (w/w) in acetone topically

Route:

epicutaneous, occlusive

Vehicle:

other: corn oil (i.d. induction) and acetone (challenge)

Concentration / amount:

Induction:
- 10% in corn oil intradermal
- undiluted topically
Challenge:
10% (w/w) in acetone topically

No. of animals per dose:

Test group: 20
Negative control group: 10

Details on study design:

RANGE FINDING TESTS:
By intradermal route: tested concentrations: 25%, 10% and 5% (w/w)
By cutaneous route
Under the conditions of the induction phase: tested concentrations: 100% and 50% (w/w)
Under the conditions of the challenge phase: tested concentrations: 100%, 50%, 25% and 10% (w/w)

Challenge controls:

The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole

Positive control results:

Mercaptobenzothiazole induced positive skin sensitization reactions in 100% (10/10) guinea pigs

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Signs of irritation during induction:
yes

Evidence of sensitisation of each challenge concentration:
No cutaneous reactions were observed after the challenge application of the test item.

Other observations:
No deaths and no clinical signs were noted during the study.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Executive summary:

The potential of BISMETHYLTHIOPROPANE (BMTP) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD guideline 406. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

. Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups),

. BMTP at the concentration of 10% in corn oil (treated group) or vehicle alone (control group),

. BMTP at the concentration of 10% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

On day 8, the animals of the treated group received a topical application of the undiluted BMTP to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle (ethanol/water (80/20, w/w) under the same experimental conditions. On day 22, all animals of both groups were challenged by a cutaneous application of BMTP at the concentration of 10% (w/w) in acetone to the right flank. BMTP was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions.Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. At the end of the study, the animals were killed without examination of internal organs.

No deaths and no clinical signs were noted during the study. No cutaneous reactions were observed after the challenge application of BMTP. Under the experimental conditions of this study and according to the maximization method of Magnusson and Kligman, BISMETHYLTHIOPROPANE did not induce delayed contact hypersensitivity in guinea pigs.

Reference 9

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA/TSCA, 40 CFR 798.4100

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Study performed before the implementation of the REACH regulation

Species:

guinea pig

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Davidson’s Mill Farm, South Brunswick, NJ, USA
- Microbiological status of animals, when known: Healthy. USDA Certified
- Age at study initiation: no data
- Weight at study initiation: 273-340 grams
- Housing: gang-caged in suspended stainless steel cages with mesh floors
- Diet: Purina Guinea Pig Pellets ad-libitum
- Water: ad libitum
- Acclimation period: 6 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature: 66-73°F

Route:

epicutaneous, semiocclusive

Vehicle:

other: 95% ethyl alcohol

Concentration / amount:

20%/0.5 mL

Day(s)/duration:

10 dose applications over 3.5 weeks

Adequacy of induction:

other: Maximum Non-irritating Dose

Route:

epicutaneous, semiocclusive

Vehicle:

other: 95% ethyl alcohol

Concentration / amount:

20%/0.5 mL

Day(s)/duration:

Fourteen days after the 10th application

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10

Challenge controls:

95% Ethyl Alchol

Positive control substance(s):

yes

Remarks:

0.08% Dinitrochlorobenzene (DNCB) in 95% ethyl alcohol

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20%/0.5 ml

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20%/0.5 ml

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 mL

No. with + reactions:

0

Total no. in group:

5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.5 mL

No. with + reactions:

0

Total no. in group:

5

Test Product - Diethyl Sulfide, Tech.: Only 1 site exhibited very faint non-confluent erythema 24 hours after challenge. All other sites were clear. By 48 hours all sites were clear. There was no evidence of edema at either time interval. Naive Control: None of the challenged sites exhibited erythema or edema 24 or 48 hours after dosing.

Positive Control (0.08% DNCB in 95% Ethyl Alcohol): Nine test sites were erythematous 24 hours after challenge. Scores ranged from 1-2 (faint confluent to moderate erythema). One site was clear. Erythema persisted through 48 hours but decreased slightly. No edema was noted at either time interval.

The DNCB naive guinea pigs had very faint to faint erythema 2¿hours after dosing. By 48 hours 3 sites cleared and 2 still exhibited very faint, non-confluent erythema.

Interpretation of results:

GHS criteria not met

Conclusions:

Diethyl Sulfide, Tech. does not cause contact sensitization when applied at its highest non-irritating dose (20% w/w in 95•ïethylalcohol) 10 times over a 3-1/2 week induction period.

Executive summary:

Diethyl sulfide, Technical, was dermally applied to guinea pigs in ten induction doses at a concentration of 20% (w/w) in 95% ethyl alcohol (highest non-irritatingdose). Fourtendays after the induction period, induced and naive guinea pigs were given a challenge dose. The naive animals exhibited no reaction and only one induced animal showed very sligth erythema 24 -hours after dosing. No contact sensitization response was noted

Reference 10

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 12 weeks old
- Weight at study initiation: 18.2 g to 20.9 g
- Housing:group of two (preliminary test) or four (main test) in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2
- Humidity (%):30 to 70
- Air changes (per hr):12
- Photoperiod (hrs dark / hrs light):12h/12h

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

100%, 50%, 25%, 10% and 5%

No. of animals per dose:

5

Details on study design:

TREATMENT
Rationale for concentration selection
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
the concentrations were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an increase of the ear thickness = 25%.

Preliminary test
Treatment groups
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed in four animals.

The treatment groups for the preliminary test are detailed in the following table:

Group Number of animals Concentration (%)
Left ear Right ear
1 2 females 25 100
2 2 females 10 50

Application
On Days 1, 2 and 3, a dose-volume of 25 µL of the appropriate dose formulation preparation was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip.
In order to avoid licking, to ensure an optimized application of the test materials and to facilitate ear thickness measurement, the animals were placed under light isoflurane anesthesia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
As the dose formulations were solutions, they were stirred manually before the dosing procedure.

Main test
Treatment groups
The treatment groups for the main test are detailed in the following table:

Group Number of animals
Treatment Concentration (%)
3 4 females Vehicle 0
4 4 females Test item 5
5 4 females Test item 10
6 4 females Test item 25
7 4 females Test item 50
8 4 females Test item 100
9 4 females 25% of HCA

Application
On Days 1, 2 and 3, a dose-volume of 25 µL of the control or dose formulation preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
As the dose formulations were solutions, they were stirred manually before the dosing procedure.

CLINICAL EXAMINATIONS
Morbidity and mortality
Each animal was checked for mortality and morbidity once a day during the acclimation, treatment and observation periods, including weekends and public holidays.

Data from mortality observations collected during the acclimation phase are not presented in the study report as no mortality and no clinical signs occurred during this period.

Clinical signs
Each animal was observed once a day from Day 1 to Day 6 (at approximately the same time each day) for the recording of clinical signs.

Body weight
The body weight of each animal was recorded once during the acclimation period, and then on the first day of treatment and on the day of sacrifice.

Ear thickness measurements and recording of local reactions
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as potent irritants may yield positive lymphoproliferative responses of equivocal significance in the LLNA.

On Days 1, 2 and 3 (before each cutaneous application) and on Day 6 (immediately after sacrifice), the thickness of both ears of each preliminary test animal and the left ear of each main test animal was measured, using a micrometer.
The increase in ear thickness was expressed as percentages between Day 1 and Day 3, and between Day 1 and Day 6, and was used to determine the irritation level due to the test item application.

No measurement of ear thickness was performed for the main test animals of the positive control group.

The irritation level of the test item was determined according to the following table:

% increase in ear thickness Irritation level Interpretation
< 10% I Non-irritant
= 10 - < 25% II Slightly irritant
= 25% III Irritant

AURICULAR LYMPH NODE CELL PROLIFERATION ASSAY
Intravenous injection of 3H-TdR and sampling of auricular lymph nodes
Lymph node cell proliferative response was measured as described by Kimber and Dearman (1). On Day 6 of the main test, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 to 21 µCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
In the following 5 h to 5 h 30 min, the main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the Auricular Lymph Nodes (ALN) were excised. Two ALN were collected per animal. ALN were then pooled for each experimental group before processing.
A single cell suspension of ALN was prepared, for each group, by mechanical disaggregation in Petri dishes using the plunger of a syringe.

Preparation of auricular lymph node cell suspensions and determination of proliferation
Cell suspensions were washed once with 15 mL of 0.9% NaCl. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic (TCA) at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three milliliters of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using ¿-scintillation counting.
The results are expressed as disintegrations per minute (dpm) per group and as dpm/node.

Stimulation Indices (SI) were calculated according to the following formula:

SI = dpm per node of the treated group / dpm per node of the vehicle control group

Acceptance criteria
The study is considered valid if the SI for the positive control is higher than the threshold positive value of 3.

Evaluation of results
The test item is considered as a skin sensitizer when the SI for a dose group is > 3 together with consideration of a dose-response relationship. Other relevant criteria such as chemical toxicity, radioactivity levels and ear thickness are also taken into account to evaluate the data.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

SI = 8.64

Parameter:

SI

Remarks on result:

other: 5 % = 0.66 10 % = 1.03 25 % = 1.88 50% = 0.99 100% = 1.68

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

Dimethyl Sulphide, tested as a solution after preparation at concentrations of 5, 10, 25, 50 and 100% in Acetone/Olive Oil (4/1; v/v), gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

Executive summary:

The potential of Dimethyl Sulphide to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted according to the OECD test guideline no. 429. A test to select the vehicle was conducted to ensure solubility of the test item in standard vehicles used in LLNA. Then, to assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item, by topical route to the dorsal surface of both ears (one concentration per ear) on Days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From Day 1 to Day 3 then on Day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and then on Days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on Days 1, 2 and 3 at concentrations of 5, 10, 25, 50 or 100% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (Acetone/Olive Oil (4/1; v/v)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, a-hexylcinnamaldehyde (HCA), at 25%in a mixture Acetone/Olive Oil (4/1; v/v) under the same experimental conditions. From Day 1 to Day 3 then on Day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on Days 1 and 6. After 2 days of resting, on Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (³H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of ³H-TdR. The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

A solution was obtained at the highest concentration of 50% in Acetone/Olive Oil (4/1; v/v). No unscheduled deaths and no clinical signs were observed during the observation period. Body weight of animals was unaffected by the test item treatment. No local reactions were observed in any animals. In all test item-treated groups, the mean increase in ear thickness was below 10%. The test item was therefore considered as non-irritant. As the acceptance limit of 25% was not reached at any concentrations, no bias in the results of proliferation measurements linked to an excessive irritant potential was considered.The SI of the positive control was > 3; this experiment was therefore considered valid. No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.

Dimethyl Sulphidegave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

Reference 11

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

issued on 30-APR-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study performed before implementation of REACH regulation.

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France.
- Age at study initiation: on day 1, the animals of the main test were 1-2 months old
- Weight at study initiation: on day 1, the animals of the main test had a mean body weight ± standard deviation of 513 ± 34 g for the males and 442 ± 26 g for the females.
- Housing: during the acclimation period and throughout the study, the animals were housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle.
- Diet: free access to 106 pelleted diet
- Water: drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.
- Acclimation period: at least 5 days before the beginning of the study.


ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 30 to 70%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: light/dark cycle 12 h/12 h


IN-LIFE DATES: From 18-DEC-2003 to 16-FEB-2004

Route:

intradermal and epicutaneous

Vehicle:

other: corn oil (intradermal injection), a mixture ethanol/water (80/20, w/w) for the induction phase (topical) and acetone for the challenge application

Concentration / amount:

induction by intradermal injection: 5%
induction by topical application: undiluted
challenge application: 50%

Route:

epicutaneous, occlusive

Vehicle:

other: corn oil (intradermal injection), a mixture ethanol/water (80/20, w/w) for the induction phase (topical) and acetone for the challenge application

Concentration / amount:

induction by intradermal injection: 5%
induction by topical application: undiluted
challenge application: 50%

No. of animals per dose:

control group: 5 males and 5 females
test group: 10 males and 10 females

Details on study design:

RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
- By intradermal route:
> tested concentrations: 10% and 5% (w/w)
> local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
- By cutaneous route and under the conditions of the induction phase:
> tested concentrations: 100% and 50% (w/w)
> cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
- By cutaneous route and under the conditions of the challenge phase:
> tested concentrations: 100% and 50% (w/w)
> cutaneous reactions were evaluated 24 and 48 hours after removal of the dressings.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6 intradermal injection on Day 1 and 1 topical application on Day 8
- Exposure period: once for the intradermal injections and 48h for the topical application
- Test groups:
> On Day 1, three injections of 0.1 mL were made into each side of this interscapular region:
- FCA at 50% (v/v) in 0.9% NaCl
- test item at 5% (w/w) in corn oil
- test item at 5% (w/w) in the mixture FCA/0.9% NaCl (50/50)
> On Day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the undiluted test item
(As the test item was shown to be irritant during the preliminary test, a topical application of sodium lauryl sulfate was not necessary on day 7)
- Control group:
> On Day 1, three injections of 0.1 mL were made into each side of this interscapular region:
- FCA at 50% (v/v) in 0.9% NaCl
- corn oil
- vehicle at 50% (w/v) in a mixture FCA/0.9% NaCl (50/50)
> On Day 8, the animals of the control group received an application of the vehicle alone
- Site: interscapular area
- Frequency of applications: once on day 1 and on day 8
- Duration: from day 1 to day 8 of the study
- Concentrations: 5% for the intradermal injection and undiluted for the topical application


B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: day 22
- Exposure period: 24 hours
- Test groups: 50% methyl ethyl sulfide in acetone (right flank) and vehicle alone (left flank)
- Control group: 50% methyl ethyl sulfide in acetone (right flank) and vehicle alone (left flank)
- Site: posterior right and left flanks
- Concentrations: 50% methyl ethyl sulfide
- Evaluation: 24 and 48 hours after removal of the dressing of the challenge application

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (CIT study No. 26182 RDG - September 2003)

Positive control results:

In a recent study performed under CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 100% animals.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

No systemic clinical signs and no deaths were noted during the study

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: No systemic clinical signs and no deaths were noted during the study.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

No systemic clinical signs and no deaths were noted during the study

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: No systemic clinical signs and no deaths were noted during the study.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the maximization method of Magnusson and Kligman, the test item METHYL ETHYL SULFIDE should not be considered as a skin sensitizer.

Executive summary:

The potential of the test item METHYL ETHYL SULFIDE to induce delayed contact hypersensitivity was evaluated in guinea pigs according OECD testing guideline 406 and GLP.

30 guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females.

On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

- Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups),

- test item at the concentration of 5% in corn oil (treated group) or vehicle alone (control group),

- test item at the concentration of 5% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

On day 8, the animals of the treated group received a topical application of the undiluted test item to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions.

On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 50% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

No systemic clinical signs and no deaths were noted during the study.

After the challenge application, no cutaneous reactions were observed in the animals of the control group. In the treated group, a discrete erythema was noted on the right treated flank of 1/20 animals at the 24 and 48-hour readings. A discrete erythema was also recorded on the left control flank of 1/20 animals, at the 24-hour reading only.

The persistent cutaneous reactions observed in 1/20 animals of the treated group may be attributable to delayed contact hypersensitivity. However, as this possible positive reaction occurred in a single animal, the test item should not be considered as a skin sensitizer.

Reference 12

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD SIDS

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Test performed before the implemenation of the REACH regulation

Species:

guinea pig

Sex:

male/female

Route:

intradermal

Vehicle:

water

Concentration / amount:

2%

Day(s)/duration:

Day 1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.3 ml

Day(s)/duration:

Day 8

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.1 mL

Day(s)/duration:

Day 22

No. of animals per dose:

10

Positive control substance(s):

not specified

Hours after challenge:

24

Group:

test chemical

Dose level:

0.1 mL

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1 mL

No. with + reactions:

0

Total no. in group:

20

Interpretation of results:

GHS criteria not met

Executive summary:

A group of 10 male and 10 female guinea pigs were induced intradermally using 2% m/v sulfolane in water/Freunds Complete Adjuvant followed a week later by topical induction using undiluted sulfolane (0.3 ml) which was applied over the sites of the intradermal injections and covered occlusively for 48 hours. Challenge was carried out 3 weeks after the intradermal induction. Undiluted sulfolane (0.1ml) was applied to the shaven backs of the test animals and covered with occlusive tape for 24 hours. Dermal reaction to the challenge was assessed after removal of the bandages and at 24 hours and 48 hours after challenge.

None of the twenty test animals showed any positive response at either 24 or 48 hours after removal of the challenge patches. It may be concluded therefore, that the substance is not a skin sensitiser in guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with Regulation (EC) No 1272/2008, no classification is warranted for the skin sensitization of tetrahydrothiophene.