1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08/02/2018 - 06/04/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

see principles of method below for full details

Principles of method if other than guideline:

Deviations from the guidelines:
- The standard states MTT must be used for an incubation period of 4 hours, solubilised overnight in 10% SDS and read at 690 nm. The study uses MTT for a 3 hr incubation period, which is solubilised in isopropanol and read at 570 nm.
- The standard states that cinnamic aldehyde must be used at 4 - 64 μM, whereas the study uses cinnamic aldehyde 8 - 128 μM.
Neither deviations are considered to affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The test (KeratinoSens) 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) and is considered scientifically valid when used as part of an integrated approach to testing to determine skin sensitisers and non-sensitisers for the purposes of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

EC Number:

268-761-3

EC Name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Cas Number:

68139-30-0

Molecular formula:

C9H19N2O5S(CnH(2n+1)), (n=7,9,11,13,15,17)

IUPAC Name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DSP-001
- Expiration date of the lot/batch: 15th August 2018
- Purity test date: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Assumed to be stable
- Stability under test conditions:Assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: Assumed to be stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Assumed to be stable

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Using a test system (KeratinoSens cell line derived from the HaCaT human keratinocytes) a luciferase reporter gene transcriptionally controlled by the Anti-oxidant Response Element from a gene that is known to be up regulated in contact with sensitisers was quantitatively measured.
Experiments were performed in plates where cells were exposed in individual concentrations to the test substance and appropriate control substances.
Solubility of the test item in cell culture medium was confirmed up to 200 mg/ml. Subsequent dilution in cell culture medium with 1% ethanol yielded a top concentration of 400 µg/ml.

Results and discussion

Positive control results:

The positive control, Cinnamic aldehyde, showed that there was a dose-dependent increase of induction =>1.5-fold in at least one concentration (at both 64 and 129 μM) and the CV% of blank values was < 20%, therefore the results are considered as valid.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Test Item

Parameter:

other: EC1.5

Value:

1.5

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Test item

Parameter:

other: Imax

Value:

0.854

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Not specified.
- Acceptance criteria met for positive control: Pass.
- Acceptance criteria met for variability between replicate measurements: Not all criteria met, however results are considered valid.
- Range of historical values if different from the ones specified in the test guideline: No data.

Applicant's summary and conclusion

Interpretation of results:

other: borderline non-sensitiser

Conclusions:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts was classified as a borderline non-sensitiser based on the results of the OECD 442D in vitro skin sensitisation test.

Executive summary:

The GLP-compliant study performed according to the OECD Test Guideline 442D, assessed the in vitro sensitisation potential of 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts using the test system (KeratinoSens test method).

The experiment uses the quantification of luciferase expression in the cell reporter line to determine the response to the test item, a negative control and a positive control, reporting the EC1.5 and Imax values.

After 48h exposure of cells with 12 concentrations of the test item, luciferase measurements and MTT viability testing were performed. The test item produced luciferase induction >1.5 in all 3 repetitions. The respective EC1.5 values were calculated as 16.48 µg/ml for repetition 1; 7.63 µg/ml for repetition 2 and 2.5 µg/ml for repetition 3. However, no dose response was observed in Rep1 and the viability was less than 70% in Rep 3 at the dose that elicited the induction above the threshold.