Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-02-14 to 2018-03-14

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

IUPAC Name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Test material form:

other: solid, very viscous yellow paste

In chemico test system

Details on the study design:

TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM test item solutions
- Vehicle: water

CONTROLS
- Solvent controls:
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide)
- Co-elution control:
- Sample prepared from the respective peptide buffer and the test item, but without peptide
- Positive control:
-- Cinnamaldehyde was used as 100 mM solution in acetonitrile for the Cys-peptide.
-- 2,3-Butanedione was used as 100 mM solution in acetonitrile for the Lys-peptide

PEPTIDES
- Cys-Peptide (Cysteine): Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
- Lys-Peptide (Lysine) Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
- Stock solution:
-- 0.667 mM Cys-Peptide solution was prepared by dissolving 22.6 mg of the peptide in 45 mL phosphate buffer, pH 7.5.
-- 0.667 mM Lys-Peptide solution is prepared by dissolving 23.3 mg of the peptide in 45.0 mL ammonium acetate buffer, pH 10.2. (Test 1)

EXPERIMENTAL PROCEDURE
- Two valid experiments were performed (first experiment and verification)
- Replicates: 3 for each peptide
- The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h
- Determination remaining non-depleted peptide concentration: HPLC at 220 nm
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration standards were prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM peptide
- The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions. A final volume of 1 mL per sample was prepared for each sample.

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC system from Agilent Technologies
- Wavelength: 220 nm and 258 nm
- Detector: UV/VIS-Detector DAD G1315A

DATA EVALUATION
The peptide depletion was calculated for each individual sample using the following equations (shown for Cys-Peptide, Lys-peptide is calculated analogously):

- Peptide depletion C,i = (1-measured peptide peak area in sample/mean peptide peak area in Solvent controls C ) × 100 %

The mean peptide depletion of the Cys-peptide was calculated as follows:

- Peptide depletion Cys = ∑Cys,i = 1,2,3 Peptide depletion i / 3

The mean peptide depletion of the test item was calculated using the following equation:

- Mean peptide depletion [%] = Peptide depletion Cys [%] + Peptide depletion Lys [%] / 2

ACCEPTANCE CRITERIA
- The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide
- The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide
- The maximum standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion

EVALUATION RESULTS
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.

Results and discussion

Positive control results:

The mean peptide depletion values for the positive controls were fullfilled

In vitro / in chemico

Results

Results 1

Run / experiment:

other: first experiment

Parameter:

other: Cys-peptide depletion [%]

Value:

7.15

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: first experiment

Parameter:

other: Lys-Peptide depletion [%]

Value:

1.54

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: verification

Parameter:

other: Cys-peptide depletion [%]

Value:

11.56

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Run / experiment:

other: verification

Parameter:

other: Lys-Peptide depletion [%]

Value:

0.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 5

Run / experiment:

other: Mean first experiment

Parameter:

other: Mean peptide depletion [%]

Value:

4.35

Results 6

Run / experiment:

other: Mean verification

Parameter:

other: Mean peptide depletion [%]

Value:

6.03

Other effects / acceptance of results:

All acceptance criteria were fulfilled

Any other information on results incl. tables

Calculated peptide depletion values for the Lys-Peptide, first experiment:

Sample	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)
Positive control	31.30	32.76	1.548
	32.52
	34.44
Test item	0.59	1.54	2.18
	0.00
	4.04

Calculated peptide depletion values for the Cys-Peptide, first experiment:

Sample	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)
Positive control	74.78	77.61	2.46
	78.77
	79.27
Test item	2.80	7.15	4.12
	7.65
	10.99

Calculated peptide depletion values for the Lys-Peptide, verification experiment:

Sample	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)
Positive control	24.28	26.01	2.69
	24.64
	29.11
Test item	1.39	0.50	0.77
	0.00
	0.10

Calculated peptide depletion values for the Cys-Peptide, verification experiment:

Sample	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)
Positive control	79.37	81.40	2.00
	81.47
	83.37
Test item	8.58	11.56	2.92
	11.69
	14.42

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

The study alone cannot be used for the classification purpose but in a WoE approach of minimally 2 in vitro assessment representing the key events in AOP

Conclusions:

The substance showed minimal reactivity in the DPRA study. The substance was found to be “negative” with minimal reactivity.

Executive summary:

In the current study the skin sensitisation effect of the test item was assessed in an in chemico assay according to OECD 442C and in compliance to GLP. The study was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. Two valid experiments were performed, whereby experiment 2 was conducted in order to verify the results of experiment 1. In both experiments a 100 mM test item solution in water was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 4.35 % in experiment 1, therefore the test had to be repeated for verification of this result. The mean peptide depletion in the Lys-peptide and Cys-peptide in the repeated experiment was 6.03 %. The result of the first experiment was considered verified, because the classification is “negative” for both experiments. All acceptance criteria were fulfilled and the study is valid. The DPRA predicition is “negative” with minimal reactivity and can be considered as non-sensitiser.