Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Skin sensitisation
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
Materials and methods
Test guideline
- 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.
Test material
- Reference substance name:
- N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride
- Molecular formula:
- C15H23ClN2O
- IUPAC Name:
- N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2-enoyl]amino}propan-1-aminium chloride
- Test material form:
- other: Deliquescent solid
- Details on test material:
- batch/lot number: PS-196-943
Expiry: 23rd November 2022
Constituent 1
- Specific details on test material used for the study:
- Storage: room temperature
Supplier: sponsor
batch/lot number: PS-196-943
Expiry: 23rd November 2022
In vitro test system
- 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.
Results and discussion
- 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.
In vitro / in chemico
Resultsopen allclose all
- 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
Applicant's summary and conclusion
- 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.
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