Trimethylsulfonium bromide

Endpoint summary

Description of key information

The test item is considered as not sensitiser in both the in chemico direct peptide reactivity assay (DPRA) and the in vitro KeratinoSens™ assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 26 October to 12 November, 2021

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 KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Lot No.: 21MR1-2
Purity: 99%

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution:
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dist. water. A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube. A 0.2 µm sterile filter was used to sterilise the test sample solution. Vortex mixing and sonication were used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. Since the test item was dissolved in water, DMSO was added at a final concentration of 4% (v/v).
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

- Preparation of the positive controls:
Cinnamic aldehyde was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

- Preparation of the solvent, vehicle and negative controls:
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.


DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 uM
- Cytotoxicity assessment performed: yes
- Final concentration range selected on basis of: Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 2
- Test chemical concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
- Application procedure
- Exposure time: 48 hours
- Study evaluation and decision criteria used:
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 1000 µM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with test items at a maximal test concentration < 1000 µM and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration is considered as inconclusive.

LUCIFERASE ACTIVITY MEASUREMENTS
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

DATA EVALUATION
- Cytotoxicity assessment:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Vehicle / solvent control:

water

Negative control:

other: DMSO

Positive control:

cinnamic aldehyde [442D]

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Cell viability:

No cytotoxic effects were observed in the tested concentration range.

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no EC1.5 value could be calculated.

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No cytotoxic effects were observed in the tested concentration range.
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No cytotoxic effects were observed in the tested concentration range.
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as non-sensitiser.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the condition of this study the test item is therefore considered as non-sensitiser.

Executive summary:

The in vitro KeratinoSens™ assay was performed according to OECD Guideline 442D under GLP.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 19 October to 02 December, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Lot No.: 21MR1-2
Purity: 99%

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
20.74 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (40.04 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
20.42 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (40.0 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

- Preparation of the test chemical solutions:
The test item was pre-weighed into a glass vial and was dissolved in water. A stock solution with a concentration of 100 mM was prepared.

- Preparation of the positive controls, reference controls and co-elution controls:
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides. Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

INCUBATION
- Incubation conditions: The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.
- Precipitation noted

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range.
- Verification of the suitability of the HPLC for test chemical and control substances

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm, 258 nm

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 69.42%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0.29 %

At concentration:

0.5 mM

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0.34 %

At concentration:

0.5 mM

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cwater).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.31%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) was performed according to OECD Guideline 442C under GLP.

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The in chemico direct peptide reactivity assay (DPRA) was performed according to OECD Guideline 442C under GLP. In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.

The in vitro KeratinoSens™ assay was performed according to OECD Guideline 442D under GLP. In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation:

DPRA, OECD 442C: negative;

KeratinoSens OECD 442D: negative.

According to Regulation (EC) No 1272/2008, section 3.4, this substance should not be classified for this endpoint.