Ethyl lactate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

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)

Justification for non-LLNA method:

The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential.
To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC.
The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl lactate

EC Number:

202-598-0

EC Name:

Ethyl lactate

Cas Number:

97-64-3

Molecular formula:

C5H10O3

IUPAC Name:

ethyl 2-hydroxypropanoate

Test material form:

liquid

Remarks:

colorless transparent liquid

In vitro test system

Details on the study design:

6.2.1 HPLC system
Designation: HPLC_4
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981
Usage and calibration followed the corresponding SOP 114 00 526 in the current edition.
6.2.2 Column
An ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phe-nomenexSecurityGuard C18, 4x3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline.
6.2.3 HPLC program
Eluent A H2O + 0.1 % TFA
Eluent B Acetonitrile + 0.085 % TFA
Gradient time (min) % A % B
0 90 10
10 75 25
10.5 10 90
12 10 90
13 90 10
20 90 10
Flow rate 0.55 mL/min
Injection volume 7 µL
Column temperature 30 °C
Wavelength 1 220 nm
Wavelength 2 258 nm

6.3 Test System
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
6.3.1 Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.:P170415-2-LR569638
Purity: 98.10%
6.3.2 Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170415-2-LR569640
Purity: 98.85%

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Cys-Peptide

Parameter:

other: Depletion [%]

Value:

3.39

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Results 2

Run / experiment:

other: Lys-Peptide

Parameter:

other: Depletion [%]

Value:

0.16

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

DPRA Prediction: Negative

Reactivity class: Minimal

Applicant's summary and conclusion

Conclusions:

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 1.77 %, there-fore the test item was classified with:

DPRA Prediction: Negative
Reactivity class: Minimal