[No public or meaningful name is available]

Endpoint summary

Description of key information

The human skin sensitisation potential of the test substance was assessed using the validated in vitro method, the KeratinoSensTM assay. After 48±2h exposure of cells with 12 concentrations (in the range 0.977 to 2000 µM)  of the test substance, luciferase measurements and MTT viability testing were performed. Cinnamic Aldehyde as a positive control and DMSO as a negative control were used. Five repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=15 overall) and 2 x 96-well plates for MTT (n=10 overall). The validity of each repetition was assessed following acceptance criteria. The test item was classified based on the concordance of Repetitions 1 and 4 (based on a 2 out of 3 approach). In this study, the test substance was classified as a Negative using the KeratinoSens prediction model.

The human skin sensitisation potential of the test substance was assessed using the validated in vitro method, OECD TG 442E, the h_CLAT (human Cell LineActivation Test) assay. For the expression measurements, test concentrations in a range from 1239.20 to 4440.28 μg/mL (after dilution in medium) were used. Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10⁶ cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry. In both experiments the RFI values for CD86 were <150% and the RFI values for CD54 were <200%. The test article therefore gave a negative prediction in the assay.All acceptance criteria of the h-CLAT assay parameters were met in each experiment. The positive control was 2,4-dinitrochlorobenzene (DNCB) prepared in anhydrous dimethyl sulphoxide (DMSO) (2 mg/mL stock), diluted in culture medium to obtain a working solution of 8 µg/mL, and final treatment concentration of 4 µg/mL in the plate. Treatments with the positive control solvent DMSO were also included in the assay.

In addition, the skin sensitising potential of the substance was evaluated by obtaining predictions using the automated workflow in the OECD QSAR Toolbox after a review of the literature did not identify usable experimental data. As alerts were returned from the initial profiling, predictions were made from Guinea Pig Maximisation Test and the Local Lymph Node Assay (LLNA) with substances leading to Michael addition upon protein binding according to the "Protein binding alerts for skin sensitisation by OASIS" profiler. The result of the prediction was negative. It was therefore concluded that the substance is not expected to induce skin sensitisation.

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:

key study

Study period:

19/07/2022 - 13/09/2022

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)).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: THP-1 cell surface marker expression

Justification for non-LLNA method:

The test method (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Specific details on test material used for the study:

Storage: room temperature
Supplier: sponsor
batch/lot number: PS-196-943
Expiry: 23rd November 2022

Details on the study design:

Skin sensitisation (In vitro test system)
- Details on study design:
THP-1 cells were pre-cultured for either 48 or 72 hrs. Following this, the cells were dosed with the test item over an 8 dose range and incubated for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test item that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs.
THP-1 cells were pre-cultured again for 48hrs. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test item. This dilution range was used to dose the cells again for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.

Positive control results:

The positive control was 2,4-dinitrochlorobenzene (DNCB) prepared in anhydrous dimethyl sulphoxide (DMSO) (2 mg/mL stock), diluted in culture medium to obtain a working solution of 8 µg/mL, and final treatment concentration of 4 µg/mL in the plate.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

Treatments with the positive control solvent DMSO were also included in the assay.
The solvent/vehicle control was culture medium (as defined in the Guideline for test chemicals solubilised or stably dispersed in medium or saline).
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.

Run / experiment:

other: 1

Parameter:

other: CD54 RFI

Value:

107

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 RFI

Value:

77

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: CD54 RFI

Value:

136

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 RFI

Value:

86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

The RFI values for CD54 and CD86 expression did not cross the sensitisation thresholds, therefore, N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was classified as a non-sensitiser as per the prediction model.

Executive summary:

The study assesses the in vitro sensitisation potential of N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride using the test system (h-CLAT test method). The study is GLP compliant performed according to the OECD Guideline 442E. The experiment uses the quantification of cytotoxic effects observed (CV75 assay) and cell surface marker expression on THP-1 cells (CD54 and CD86 assays) after 24-hour exposure to the test substance to determine the response. The relative fluorescence intensity (RFI) is used a measure of expression of CD54 and CD86, calculated from the results at test item doses in duplicate.

The expression of CD54, as measured by the RFI, did not cross the threshold (RFI ≥200) at any dose.

The expression of CD86, as measured by the RFI, did not cross the threshold (RFI ≥150) at any dose.

Cell viability did not fall below 50% at any of the test item concentrations and therefore the result is deemed to be valid.

Therefore, under the conditions of this test N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride is not considered a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 Feb to 12 Mar 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)

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values. The adapted method showed full concordance with the Validated Reference Method (VRM) – the KeratinoSensTM standard protocol. XCellR8 recently obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, are included in the dossier (Annex 3).

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

Method of administration of test item:
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing

Method of administration of reference items:
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).

Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h prior to endpoint measurements.

Number of repetitions:
Five repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=15 overall) and 2 x 96-well plates for MTT (n=10 overall). The validity of each repetition was assessed following acceptance criteria described in section 12.1.

Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Cell seeding (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells
per well, passage number 18 for repetitions 1 to 3, passage number 20 for repetitions 4 to 5.
Day 2: 24 hours after seeding, the test and control items were applied and plates were incubated at
37oC, 5% CO2, ≥ 95% relative humidity for 48 ± 2 hours.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing
(2 plates)

Solubility Assessment:
The test concentrations of N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride used in the KeratinoSensTM method were selected on the basis of solubility test carried out during the study.
Solubility of the test item in was confirmed up to 200mM in DMSO. Subsequent dilution in cell culture medium gave a top concentration of 2000µM.

Negative control:

other: DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Assay Acceptance Criteria

Test results are acceptable if:
- The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction produced by this control is above the threshold of 1.5 in at least one of the tested concentrations and this induction is statistically significant compared to the solvent (negative) control (p<0.05).
- The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets:
Average induction in the three replicates for cinnamic aldehyde at 32 µM is within the XCellR8 historical range (currently 1.6 and 3)
EC1.5 value for cinnamic aldehyde is within the XCellR8 historical range (currently 6 µM and 39 µM).

Note: At least one of these criteria must be met, otherwise the run is discarded unless there is sufficient reason not to do this as determined by the Study Director.
If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
CV% of blank values < 20%

Interpretation of Results and Skin Sensitisation Prediction model

A test item is considered positive using the KeratinoSens prediction model if the following conditions are met in 2 of 3 repetitions:

The IMAX is ≥ 1.5-fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test).

The cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity ≥ 1.5-fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.

The EC1.5 value is < 1000 µM or < 200 µg/mL for test items with no defined MW.

There is an apparent overall dose-response for luciferase induction (or a biphasic response).

Key result

Group:

test chemical

Run / experiment:

other: 4

Parameter:

EC 1.5 [442D]

Value:

1 447.843 µM

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: 4

Parameter:

Imax [442D]

Value:

1.63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: The test item was classified based on the concordance of Repetitions 1 and 4 (based on a 2 out of 3 approach).

See result tables attached

Interpretation of results:

GHS criteria not met

Conclusions:

N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was tested in the KeratinoSens prediction model with negative results. Classification as a skin sensitiser is subsequently not required (CLP Regulation (EC) No 1907/2006).

Executive summary:

The human skin sensitisation potential of N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was assessed using the validated in vitro method, the KeratinoSensTM assay, adapted to animal product-free conditions by XCellR8, and validated in-house to determine keratinocyte activation.

After 48±2h exposure of cells with 12 concentrations (in the range 0.977 to 2000 µM)  of N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride, Luciferase measurements and MTT viability testing were performed. Cinnamic Aldehyde as a positive control and DMSO as a negative control were used.

Five repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=15 overall) and 2 x 96-well plates for MTT (n=10 overall). The validity of each repetition was assessed following acceptance criteria. The test item was classified based on the concordance of Repetitions 1 and 4 (based on a 2 out of 3 approach).

In this study, N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride was classified as a Negative using the KeratinoSens prediction model.

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

other: Expert Assessment

Adequacy of study:

weight of evidence

Study period:

August 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Results based on structural alert and QSAR models

Qualifier:

no guideline followed

Principles of method if other than guideline:

An expert assessment was performed using the OECD QSAR Toolbox. The OECD QSAR Toolbox is outlined by ECHA as one of the in silico tools to reliably predict skin sensitisation; it
uses read-across and numerous databases of experimental results to enable the user to fill in data gaps in a logical workflow. Relevant information for the target substance, including probable mechanisms of action and observed/simulated metabolites, is retrieved by profiling to contribute to data gathering and the identification of suitable analogues, and by grouping substances into meaningful categories according to structural similarities.

GLP compliance:

no

Type of study:

other: Expert Assessment

Key result

Group:

test chemical

Run / experiment:

other: N/A

Parameter:

other: QSAR prediction

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The prediction is based on 5 values, 3 of them (60.0%) equal to the predicted value.
Prediction confidence measured by the p-value: 0.5

As alerts were returned from the initial profiling, predictions were made from Guinea Pig Maximisation Test and the Local Lymph Node Assay (LLNA) with substances leading to Michael addition upon protein binding according to the "Protein binding alerts for skin sensitisation by OASIS" profiler.

The result of the prediction was negative.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not expected to induce skin sensitisation.

Executive summary:

The skin sensitising potential of the substance was evaluated by obtaining predictions using the automated workflow in the OECD QSAR Toolbox after a review of the literature did not identify usable experimental data.

As alerts were returned from the initial profiling, predictions were made from Guinea Pig Maximisation Test and the Local Lymph Node Assay (LLNA) with substances leading to Michael addition upon protein binding according to the "Protein binding alerts for skin sensitisation by OASIS" profiler. The result of the prediction was negative. 

It was therefore concluded that the substance is not  expected to induce skin sensitisation.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test substance was tested in the KeratinoSens prediction model with negative results.  The test substance was tested in the h-CLAT prediction model with negative results. In addtion, the result of the QSAR prediction was negative. Classification as a skin sensitiser is subsequently not required (CLP Regulation (EC) No 1907/2006).