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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2017 - 18 December 2017
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))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2,4-dichlorophenyl)ethan-1-one
EC Number:
218-780-8
EC Name:
1-(2,4-dichlorophenyl)ethan-1-one
Cas Number:
2234-16-4
Molecular formula:
C8H6Cl2O
IUPAC Name:
1-(2,4-dichlorophenyl)ethan-1-one
Test material form:
solid: crystalline

In chemico test system

Details on the study design:
Cysteine peptide
. Peptide sequence : AC-RFAACAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
. Molecular weight : 750.88 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 111016HS_MHeW1017
. Storage condition : at -20°C
. Description : White powder

Lysine peptide
. Peptide sequence : AC-RFAAKAA-COOH
. Peptide sequence synonyms : AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
. Molecular weight : 775.91 g/mol
. Supplier : JPT Peptide Technologies GmbH
. Batch No. : 220114HSDWW0117
. Storage condition : at -20°C
. Description : White powder

Vehicle:
Based on solubility results, the retained vehicle was acetonitrile without sonication step.

Test item formulation preparation:
It was dissolved in the selected vehicle (acetonitrile) at 100 Mm (without sonication step).

Positive control: Cinnamaldehyde.

Co-elution control samples:
In order to detect co-elution of the test item with a peptide, co-elution samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Chromatograms of the co-elution control samples were analyzed and compared with those of the reference control C samples.

Reference control samples:
All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent:
*reference control A: check the accuracy of the calibration curve for peptide quantification,
*reference control B: check the stability of the peptide during analysis,
*reference control C: check that the solvent did not impact the percentage of peptide depletion.

The test item was tested in one run.

INCUBATION OF THE SAMPLES:
All samples (co-elution controls, reference controls, test item and positive control samples) were incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation. Samples presenting precipitate or phase separation (micelles) were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

PREPARATION OF THE CALIBRATION CURVE SAMPLES
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also
included in the standard calibration curve. The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

HPLC/UV ANALYSIS
The study samples were assayed in batches using HPLC/UV analysis.
Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm, (Waters). In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phase: Mobile phase A: acetonitrile + 0.085% TFA; Mobile phase B: milli-Q water + 0.1% TFA
Flow: 350 μL/minute
UV Wavelength: 220 nm

CALCULATION OF THE PERCENT PEPTIDE DEPLETION
% depletion = [1 - (Peptide peak area in replicate injection / Mean peptide peak area (3 replicates) in relevant reference control samples] x 100


Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: Mean percent depletion value for cysteine peptide
Value:
0.76
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: Mean percent depletion value for lysine peptide
Value:
2.66
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. The mean percent depletion values for cysteine and lysine peptides were 0.76% and 2.66% respectively. The mean of the percent cysteine and percent lysine depletions was equal to 1.71%. Accordingly, the test item was considered to have no or minimal peptide reactivity though with limitations due to its precipitation or phase separation with the lysine peptide. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls:
The mean peptide concentrations of the reference control A samples was within ± 10% of the nominal concentration. The CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile were 0.4% for cysteine and 1.6 for lysine (i.e. < 15.0%).

- Acceptance criteria met for positive control:
The cinnamaldehyde mean percent depletion for the cysteine peptide was 75.55 with a % CV of 0.4 (i.e. between 60.8 and 100%, with a SD < 14.9%). The cinnamaldehyde mean percent depletion for the lysine peptide was 62.76 with a % CV of 7.5 (i.e. between 60.8 and 100%, with a SD < 14.9%).

- Acceptance criteria met for the calibration curve samples:
The calibration curves had a coefficient of determination (r2) ≥ 0.99

- Acceptance criteria met for variability between replicate measurements:
The mean peptide concentrations of the reference control C samples prepared in acetonitrile was within ± 10% of the nominal concentration. The maximum SD for the test item replicates was < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

OTHER: PRECIPITATION:
At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis. As precipitate and phase separation (micelles) were observed in the test item samples incubated with the lysine peptides and as phase separation (micelles) were observed in co-elution samples prepared with the lysine dilution buffer, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Positive samples incubated with both peptides were also centrifuged at the same conditions to force precipitate to the bottom of the vials. Only supernatants were then injected into the HPLC/UV system. For the other samples (i.e. all reference controls, test item and co-elution controls incubated with the cysteine peptide), the vials were directly transferred into the HPLC/UV system.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation or phase separation with the lysine peptide. The DPRA prediction is considered as negative.
Executive summary:

In chemico skin sensitization direct peptide reactivity assay (DPRA) was performed accordin to the OECD Guideline 442C (GLP study). The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent). The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. The mean percent depletion values for cysteine and lysine peptides were 0.76% and 2.66% respectively, being the mean value equal to 1.71%. Accordingly, the test item was considered to have no or minimal peptide reactivity though with limitations due to its precipitation or phase separation with the lysine peptide. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

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