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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: EU-Method B.59
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Direct Peptide Reactivity Assay
Justification for non-LLNA method:
The DPRA is part of a tiered strategy for the evaluation of skin sensitisation potential in the context of an integrated approach to testing and assessment (IATA).

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)
EC Number:
695-938-6
Cas Number:
678966-16-0
Molecular formula:
F2C4PLiO8
IUPAC Name:
Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)

In vitro test system

Details on the study design:
6.2.1 HPLC system
Designation: HPLC_8
Components: Solvent Rack SRD3400
Quaternary pump HPG-3400SD
Autosampler WPS-3000SL
Column compartment TCC-3000SD
Photometer VWD-3400RS
Manufacturer: Thermo Fisher Scientific
Software: CHROMELEON 6.80 SR15b Build 4981
Usage and calibration followed the corresponding SOP 114 00 529, edition 2 valid from 20. Sep. 2018.

6.2.2 Column
An ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phenom-enex SecurityGuard 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 guideline OECD 442C.
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 RS synthesis, (Louisville, KY, USA), were used.
6.3.1 Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.: P181203-LC180433
Purity: 96.93 %
6.3.2 Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170906-17-LC107617
Purity: 98.21 %

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
cysteine depletion
Value:
91.25 %
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
lysine depletion
Value:
14.93 %
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Other effects / acceptance of results:
DPRA Prediction: Positive
Reactivity class: High

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The DPRA prediction is ”positive” according to the Cysteine 1:10/Lysine 1:50 prediction model. Thus, under the experimental conditions reported, the test item Lithium difluorobis(oxalate)phosphate shows a reactivity towards the two model synthetic peptides.
This assignment supports the discrimination between skin sensitisers and non-sensitisers in the framework of an integrated approach (IATA).
For sensitising potency assessment within an IATA, the test item Lithium difluorobis(oxalate)phosphate could be assigned to the reactivity class that covers high reactivity under the conditions of this study.

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