[No public or meaningful name is available]

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:

2021

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:

other: direct peptide reactivity assay

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:

Tributyl(ethyl)phosphonium diphenyl phosphate

Cas Number:

2409816-82-4

Molecular formula:

C26H42O4P2

IUPAC Name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Test material form:

liquid

In vitro test system

Details on the study design:

3.2.1 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 following the corresponding SOP 114 00 526 in the current edition.
3.2.2 Column
An ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phe-nomenex SecurityGuard C18, 4x3 mm will be used. This column is used instead of the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline because it delivers substantially better peak shape for the peptides.
3.2.3 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


Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, are used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

The positive control, reference control sets C, and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h.
Samples which appear turbid or where precipitation is visible after the incubation period are centrifuged (benchtop centrifuge, 10 min at 400 g) and only the clear supernatant is used for measurement. Any occurrence of turbidity or precipitation is reported.

Positive control:

other: Cinnamaldehyde (CAS 104-55-2, food grade ≥ 95 %), 2,3-Butanedione (CAS 431-03-8, > 97 %)

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

7.65 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

mean

Parameter:

lysine depletion

Value:

1.82 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

 

Depletion [%]			Cysteine 1:10/lysine 1:50
Cys-Peptide	Lys-Peptide	Mean	Reactivity class	Prediction
7.65	1.82	4.74	Minimal	negative

Applicant's summary and conclusion

Conclusions:

All acceptance criteria were fulfilled, therefore the test was considered valid. The DPRA prediction for the test item tributyl(ethyl)phosphonium diphenyl phosphate was negative with reactivity class minimal according to the Cysteine 1:10/Lysine 1:50 prediction model.