Dodecyltrimethylammonium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-03-2018 to 09-03-2018

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

As a first approach, an in vitro test is preferred to an in vivo test. The three "R" (Reduce, Refine, Replace) rule and ECHA guidelines require the use of alternative methods before performing animal testing such as the LLNA-method.

Test material

Test material information

Constituent 1

Reference substance name:

Dodecyltrimethylammonium chloride

EC Number:

203-927-0

EC Name:

Dodecyltrimethylammonium chloride

Cas Number:

112-00-5

Molecular formula:

C15H34N.Cl

IUPAC Name:

N,N,N-trimethyldodecan-1-aminium chloride

impurity 1

Reference substance name:

Trimethyl(tetradecyl)ammonium chloride

EC Number:

224-958-6

EC Name:

Trimethyl(tetradecyl)ammonium chloride

Cas Number:

4574-04-3

Molecular formula:

C17H38N.Cl

IUPAC Name:

N,N,N-trimethyltetradecan-1-aminium chloride

impurity 2

Reference substance name:

Cetrimonium chloride

EC Number:

203-928-6

EC Name:

Cetrimonium chloride

Cas Number:

112-02-7

Molecular formula:

C19H42N.Cl

IUPAC Name:

N,N,N-trimethylhexadecan-1-aminium chloride

Test material form:

liquid

In chemico test system

Details on the study design:

Dilution condition: diluted at 100mM in acetonitril

TEST SYSTEM
Cysteine peptide (Ac-RFAACAA-COOH), purity > 90%
Lysine peptide (Ac-RFAAKAA-COOH), purity > 90%

REFERENCE ITEMS

Positive control: 100 mM cinnamaldehyde (CAS N°: 104-55-2), purity ≥ 95% (i.e. 5 ou 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).

Co-elution control: 100 mM test item in the appropriate buffer (cf § 5.4) (i.e. 5 ou 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).

3 references, made with 0.667 mM peptides solutions in acetonitrile or in the test item solvent, are included in each set of analysis: (i.e. 0.500 mM as final concentration in the reaction mixtures)
- Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy,
- Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time,
- Reference control C: prepared with the test item solvent in order to check its influence on the peptide stability and with acetonitrile, the positive control solvent.

PROTOCOL

#Series definition
The test item is prepared at 100 mM or at the maximum concentration which it can be solubilized in the appropriate solvent, as defined in § 5.2.
Positive control is prepared at 100 mM in acetonitrile.
The test item and the positive control are incubated in excess with the peptides at 1:10 and 1:50 ratio for cysteine and lysine peptides respectively.

Each sample is tested 3 times from 3 independent solutions.

#HPLC analysis
After the equilibration phase, 7 µl of each sample are automatically injected and the HPLC analysis is performed with a flow of 0.40 ml per minute
A complete sequence is performed for each sample.
The column is re-equilibrated to initial conditions (90% Phase A and 10% of phase B) at least 4 minutes between each injection.

#Sequence of the analysis
The analysis is programmed according to the number of samples and may vary depending on the number and type of samples (according to diluents used for example).
However, the following principles are respected for all sequences of analyses:
- The standards of the calibration curve and the reference controls A are placed at the beginning of the sequence.
- The reference controls B are placed at the beginning and at the end of the analysis (3 repetitions).
- The reference controls C are placed at the beginning of each repetition.

Lysine and cysteine analysis will be conducted on separate day and test item will be freshly prepared for both assays on each day. The analysis is timed to assure that the injection of the first sample starts 22 to 26 hours after the test item is mixed with the peptide solution. The HPLC analysis time will be less than 30 hours.

RESULTS

#Results expression
Results are processed by the Empower 3 software according to the current working instruction IL MAT 25.
The depletion rate is calculated for each type of peptide according to the following formula:

"Depletion % =" ("1-" "Peptide peak area with the replicate injection" /"Mean peptide peak area with the reference control C " )"×100"

Then, the mean depletion percentage for cysteine and lysine peptides is calculated for the test item and the positive control.

TEST VALIDATION

The following criteria must be checked in order to validate the test.

#Calibration curve:
Standard curve should have a coefficient r2 higher than 0.99.

#Reference control A:
The mean concentration of the peptide must be equal to 0.500 ± 0.05 mM

#Reference controls B and C:
The CV of the 9 controls B and C must be less than 15%.

#Positive Control (cinnamaldehyde):
The standard deviation of measurements must be lower than 14.9% and 11.6% for cysteine and lysine peptides respectively. The depletion mean rate must be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.4% for the lysine peptide.

#Test item validation criteria
Reference control C:
The mean concentration of the peptide must be equal to 0.500 ± 0.05 mM

Test item:
The standard deviation of measurements must be lower than 14.9% and 11.6% for cysteine and lysine peptides respectively.

Results and discussion

Positive control results:

Mean depletion % (Lysine and Cysteine) : 62.80%

In vitro / in chemico

Results

Key result

Run / experiment:

other: Depletion in Lysine and Cysteine Peptide %

Parameter:

other: depletion %

Value:

0.59

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Results test item:

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	0	0.05
Repetition 2	0	0.88
Repetition 3	1.22	1.42
Mean	0.41	0.78
SD (Standard Deviation)	0.71	0.69
SD Validity criteria	< 11.6%	< 14.9%

Results Positive control:

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	58.82	67.07
Repetition 2	56.87	67.39
Repetition 3	57.78	68.87
Mean	57.83	67.78
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

Reference Controls:

	Lysine Peptide	Cysteine Peptide
	Concentration (mM)	Concentration (mM)
Reference A	0.502	0.509
Reference C*	0.497	0.506
	CV %	CV %
Reference B/C	1.07	0.66

*Reference C: Positive control diluant, i.e. acetronitrile

Concentration validity criteria (mM): 0.500 +/- 0.050

CV validity criteria: < 15 %

Applicant's summary and conclusion

Interpretation of results:

other: reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Conclusions:

The sensitization of the test item is assessed from the mean depletion percentage of Lysine and Cysteine.
The test item, CAS # 112-00-5 RAW MATERIAL FOR NC ANTI-STATIC IIIA code ID-18/01078, presents a mean depletion percentage of 0.59% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Executive summary:

Title:

In chemico skin sensitization: Direct Peptide Reactivity Assay (DPRA) according to the OECD 442C guideline

Study principle:

A skin sensitizer is a substance inducing an allergic response after having been in contact with skin.

The molecular initiating event is the covalent binding of the electrophilic part of a molecule to the nucleophilic part of skin proteins.

 

DPRA is an in chemico method based on chemical reactivity of the test item with proteins.

The interaction between the molecule and lysine or cysteine rich peptides is detected with a high performance liquid chromatography (HPLC).

The remaining concentration of peptides is measured after 24 hours of incubation with the test item at 25°C. It is measured with the UV detector of the HPLC system, after gradient elution, at 220 nm.

The depletion rates of lysine and cysteine peptides are then used to distinguish the skin sensitizer and non-sensitizer so as to sort and label chemicals according to SGH.

 

The binding of the test item with proteins is only one of the key events in the adverse outcome pathway (AOP) of skin sensitization. Datas obtained are not enough to conclude on the absence of sensitizing capacity.

This test has to be cross-referenced with other information such asin-vitrotests carried-out on other steps of the AOP of skin sensitization.

This study is performedaccording to theOECDGuideline442CandProtocoln°154 ofECVAMDB-ALM.

Conclusion:

The sensitization of the test item is assessed from the mean depletion percentage of Lysine and Cysteine.

The test item, CAS # 112-00-5 RAW MATERIAL FOR NC ANTI-STATIC IIIA code ID-18/01078, presents a mean depletion percentage of 0.59% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.