1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 January 2017 - 12 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

210420-85-2

Constituent 2

Reference substance name:

N-methyldidecylamine

EC Number:

230-990-1

EC Name:

N-methyldidecylamine

Cas Number:

7396-58-9

Molecular formula:

C21H45N

IUPAC Name:

N-decyl-N-methyldecan-1-amine

Constituent 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid

Details on test material:

- Appearance: Slightly yellow gel
- Storage conditions: At room temperature; container flushed with nitrogen

Specific details on test material used for the study:

Purity correction factor: 1.081

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

- Test item preparation: A correction factor of 1.081 was used to correct for purity/composition of the test item in order to prepare a 100 mM test item solution.
Solubility of the test item was assessed before performing the DPRA assay. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay, 78.26 mg of the test substance was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1816 µL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

- Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany
- Rationale: recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Positive control: cinnamic aldehyde
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler, in the dark, and incubated at 25±2.5°C for 24±2 hours. Prior to HPLC-PDA analysis the samples were visually inspected. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1. The HPLC sequences of the cysteine and lysine reactivity assay for the test substance are presented in Table 2. The time between the first injection (= RCcysB-1 or RClysB-1) and the last injection of the sequence did not exceed 30 hours.
- Data evaluation: The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-((Peptide Peak Area in Replicate Injection (at 220 nm))/(Mean Peptide Peak Area in Reference Controls (at 220 nm)))]×100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%
Data interpretation: the results were evalutated according to the Cysteine 1:10 / Lysine 1:50 prediction model (Tabel 3). The threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser.

Results and discussion

Positive control results:

Mean percent SPCC depletion: 77.9% ± 1.3% (SD: 1.3%)
Mean percent SPCL depletion: 46.6% ± 3.3% (SD: 3.3%)

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: SPCC mean percentage

Value:

4.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: 4.0%

Results 2

Key result

Parameter:

other: SPLC mean percentage

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: 0.9%

Any other information on results incl. tables

Table 4 Acceptability of the DPRA assay

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.996	>0.99	0.992
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.524 ± 0.011	0.50 ± 0.05	0.500 ± 0.020
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.537 ± 0.006	0.50 ± 0.05	0.503 ± 0.011
CV (%) for RC samples B and C	<15.0	3.4	<15.0	2.4
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	77.9	40.2-69.0	46.6
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.3	<11.6	3.3
SD of peptide depletion for  1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) (%)	<14.9	4.0	<11.6	0.9

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in chemico skin sensitisation test, performed according to OECD guideline 422C and GLP principles, 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) was negative in the Direct Peptide Reactivity Assay (DPRA). Based on these reults, 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.