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Skin sensitisation

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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

Constituent 1
Chemical structure
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

Resultsopen allclose all
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
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
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.

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