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Toxicological information

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:
29 Aug - 29 Nov 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:
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:
5 Feb 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:
N-2-hydroxyethyllactamide
EC Number:
226-546-1
EC Name:
N-2-hydroxyethyllactamide
Cas Number:
5422-34-4
Molecular formula:
C5H11NO3
IUPAC Name:
N-2-hydroxyethyllactamide

In chemico test system

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

TEST SYSTEM
Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol), Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol); containing phenylalanine to aid in detection
Supplier: GenScript, Piscataway, USA; RS Synthesis, Loisville, USA

VEHICLE CONTROL
- Substance: Acetonitrile

TEST CONCENTRATIONS:
The test substance was prepared at a 100 mM concentration. 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)

CONTROLS
Negative control: Acetonitrile as vehicle control
Positive control: Ethylene glycol dimethacrylate (EGDMA, CAS 97-90-5), 50 mM in acetonitrile
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide

NUMBER OF REPLICATES: Triplicates with each peptide

MEASUREMENT HLPC
- Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 μm with guard column SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID (Phenomenex)
- Mobile Phase: A: H2O/ACN/TFA 950/50/1 V/V/V; B: ACN/H2O/TFA 950/50/0.85 V/V/V
- Flow: 0.50 mL/min
- Gradient: time [min]: 0, 8, 8.1, 10, 10.1 and 16 min; %B: 5, 20, 90, 90, 5 and 5
- Wavelength: 220 and 258 nm
- Injection volume: 2 µL
- Software: Dionex Chromeleon
- Calibation samples: 0.534, 0.267, 0.134, 0.067, 0.033 and 0.017 mM peptide


ACCEPTANCE CRITERIA
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).
In addition the positive control should cause depletion of both peptides comparable to historic data.



Results and discussion

In vitro / in chemico

Results
Key result
Run / experiment:
other: 24 ± 2 h incubation
Parameter:
other: Mean of cysteine- and lysine-depletion [%]
Remarks:
Direct Peptide Reactivity Test (DPRA)
Value:
0.78
Vehicle controls validity:
valid
Remarks:
0.00
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
36.63
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 5: Results of DRAP: Reaction with cysteine-peptide

Reaction with cysteine-peptide peptide concentration [mM] peptide depletion [%]
  sample 1 sample 2 sample 3 mean SD sample 1 sample 2 sample 3 mean SD
Vehicle control 0.486 0.482 0.497 0.488 0.008 0.44 1.33 -1.76 0.00 1.59
Test substance 0.481 0.483 0.478 0.481 0.003 1.50 1.04 2.13 1.56 0.55
Positive control 0.240 0.221 0.219 0.227 0.012 50.77 54.65 55.13 53.52 2.39

Table 6: Results of DRAP: Reaction with cysteine-peptide

Reaction with lysine-peptide peptide concentration [mM] peptide depletion [%]
  sample 1 sample 2 sample 3 mean SD sample 1 sample 2 sample 3 mean SD
Vehicle control 0.533 0.536 0.535 0.535 0.001 0.29 -0.22 -0.07 0.00 0.26
Test substance 0.537 0.550 0.538 0.542 0.007 -0.52 -2.94 -0.71 -1.39 1.35
Positive control 0.420 0.436 0.431 0.429 0.009 21.52 18.38 19.30 19.74 1.61

Applicant's summary and conclusion

Interpretation of results:
other: not skin sensitising based on the key event "direct peptide binding assay"