N-2-hydroxyethyllactamide

Skin sensitisation

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

Test material information

Constituent 1

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"