(Z)-N-methyl-N-(1-oxo-9-octadecenyl)glycine, compound with 2,2',2''-nitrilotri(ethanol) (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 Oct 2015 - 22 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

LANDESAMT FÜR UMWELT, WASSERWIRTSCHAFT UND GEWERBEAUFSICHT, Mainz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

EC Number:

250-151-3

EC Name:

Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

Cas Number:

30364-51-3

Molecular formula:

C17H33NO3.Na

IUPAC Name:

sodium [methyl(tetradecanoyl)amino]acetate

Test material form:

liquid

In chemico test system

Details on the study design:

TEST SYSTEM:
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH

TEST SAMPLE PREPARATION:
- The test substance was prepared as a 100 mM preparation (w/v) in de-ionized water (vehicle). After short stirring the test substance was soluble in the vehicle.

CONTROLS:
- Negative control (NC): = vehicle control (VC) = de-ionized water
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA) prepared as a 50 mM preparation in de-ionized water.
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

EXPERIMENTAL PRODECURE:
- The test substance was dissolved in de-ionized water. Three samples of the test substance were incubated with each peptide. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance). Additionally, triplicates of the concurrent vehicle control were incubated with the peptides. The co-elution control was prepared in the same way as the test-substance samples described above but containing the respective peptide buffer instead of peptide. The samples were prepared in suitable tubes, capped tightly and incubated at 25 °C ± 2.5 °C in the dark for 24 +/- 2 hours. Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

MEASUREMENT OF PEPTIDE CONCENTRATIONS:
- Eluent: A: 0.1% (v/v) trifluoracetic acid (≥99%) in de-ionized water; B: 0.085% (v/v) trifluoracetic acid (≥99%) in acetonitrile

- Flow rate: 0.35 mL/min
- Wavelength: 220 and 258 nm
- HPLC: Agilent HP 1100

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Mean value of triplicate samples

Parameter:

other: Cysteine peptide depletion

Value:

-8.52

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: no indication for peptide reactivity

Results 2

Key result

Run / experiment:

other: Mean value of triplicate samples

Parameter:

other: Lysine peptide depletion

Value:

0.23

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: no indication for peptide reactivity

Results 3

Key result

Parameter:

other: Mean of both depletions

Value:

0.11

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: no indication of peptide reactivity

Other effects / acceptance of results:

ACCEPTANCE CRITERIA OF THE DPRA:
- A study is considered acceptable if the positive control causes depletion of both peptides comparable to historic data.
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be <15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD <14.9% for % cysteine depletion and <11.6% for % lysine depletion).

Any other information on results incl. tables

Table 3: Mean peptide depletions of cysteine, lysine and both peptides

Test item	Cysteine peptide		Lysine peptide		Mean of both depletions [%]
	mean depletion [%]	SD	mean depletion [%]	SD
Test substance	-8.52*	0.73	0.23	1.58	0.11
Positive control	69.60	10.41	9.47	2.77	39.54

*a negative mean depletion [%] is considered as 0 for further calculation

SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: negative for peptide reactivity

Conclusions:

Based on the observed results and applying the evaluation criteria described above the test material does not exhibit peptide reactive properties in the DPRA under the test conditions chosen.