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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:
18 December 2018 - 21 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:
2,6-dimethyloct-7-en-2-yl formate
EC Number:
246-788-1
EC Name:
2,6-dimethyloct-7-en-2-yl formate
Cas Number:
25279-09-8
Molecular formula:
C11H20O2
IUPAC Name:
2,6-dimethyloct-7-en-2-ol
Test material form:
liquid

In chemico test system

Details on the study design:
Test system:
Cysteine peptide: Supplier RS synthesis, LLC (Ac RFAACAA-COOH, Batch no. 170622, purity of 94.40%)
Lysiine peptide: Supplier RS synthesis, LLC (Ac-RFAAKAA-COOH, Batch no. 170705, purity of 92.93%)

Controls:
Positive control: 100 mM cinnamaldehyde (CAS N°: 104-55-2), purity ≥ 95% (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
Co-elution control: 100 mM test item in the appropriate buffer (cf § 7.5) (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
4 references, made with 0.667 mM peptides solutions in acetonitrile or in the test item solvent, are included in each set of analysis: (i.e. 0.500 mM as final concentration in the reaction mixtures):
-Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy
-Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time
-Reference control C: prepared with the positive control solvent.in order to check its influence on the peptide stability and with acetonitrile
-Reference control C’: prepared with purified water, the test item solvent, in order to check its influence on the peptide stability

Study course:
The test item was prepared at 100 mM in purified water and the positive control was prepared at 100 mM in acetonitrile. They were incubated in excess with the peptides at 1:10 and 1:50 ratio for cysteine and lysine peptides respectively. Each sample was tested 3 times from 3 independent solutions. The vials were capped and mixed carefully avoiding bubbling, then placed in the dark, in HPLC system sampler at 25°C ± 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.

Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end of the analysis, samples vials were checked. Solution was cloudy right after preparation and at the end of analysis but no precipitation neither phase shift was observed prior the beginning and at the end of the analysis which shows the stability of the solution

The HPLC column was installed and equilibrated at 30°C with 50% of phase A and 50% of phase B, for at least 20 minutes before use. After the equilibration phase, 7 µl of each sample are automatically injected and the HPLC analysis was performed with a flow of 0.40 ml per minute at 0, 13.5, 14 and 19 minutes. The column was re-equilibrated to initial conditions (90% Phase A and 10% of phase B) at least 4 minutes between each injection.

Sequence of analysis:
The analysis was programmed according to the following principles:
-The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
-The reference controls C were placed at the beginning of each repetition.
-TThe standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.

Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.


Results and discussion

Positive control results:
See below.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: Depletion in Lysine peptide %
Value:
1.28
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Depletion in Lysine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Depletion in Lysine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: Depletion in Cysteine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Depletion in Cysteine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Depletion in Cysteine peptide %
Value:
0.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Mean depletion in Lysine peptide % was 0.43.
Mean depletion in Cysteine peptide % was 0.04.
Mean depletion % was 0.23.

OTHER EFFECTS:
- Visible damage on test system: No coelution of the test item with the peptides has been highlighted.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
Reference A: Lysine peptide concentration (mM) = 0.509, Cysteine peptide concentration (mM) = 0.502
Reference C (positive control diluent, acetonitrile): Lysine peptide concentration (mM) = 0.503, Cysteine peptide concentration (mM) = 0.498
Reference C' (test item diluent, purified water): Lysine peptide concentration (mM) = 0.480, Cysteine peptide concentration (mM) = 0.487
Validty criteria = 0.500 ± 0.050

Reference B/C: Lysine peptide CV% = 0.51, Cysteine peptide CV% = 1.76
Validity criteria < 15%

- Acceptance criteria met for positive control:
Mean depletion in Lysine peptide % was 51.17 ± 1.29 (validity criteria 40.2-69.4)
Mean depletion in Cysteine peptide % was 68.83 ± 0.55 (validity criteria 60.8-100)

Any other information on results incl. tables

Test item results:

 

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

 

 

Repetition 1

1.28

0

 

 

Repetition 2

0

0

 

Mean Depletion %

Repetition 3

0

0.11

 

Mean

0.43

0.04

 

0.23

SD

(Standard Deviation)

0.74

0.06

 

 

SD Validity criteria

< 11.6%

< 14.9%

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed mean depletion of 0.43% for Lysine and 0.04% for Cysteine, i.e. an overall average of 0.23% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Executive summary:

An in-chemico skin sensitization test was performed according to the OECD Guideline 442 (GLP study). The principle is based on chemical reactivity of the test item with proteins. The interaction between the molecule and lysine or cysteine rich peptides was detected with a high performance liquid chromatography (HPLC). The remaining concentration of peptides was measured after 24 hours of incubation with the test item at 25°C. It was measured with the UV detector of the HPLC system, after gradient elution, at 220 nm. The depletion rates of lysine and cysteine peptides were then used to distinguish the skin sensitizer and non-sensitizer. The test item showed mean depletion of 0.43% for Lysine and 0.04% for Cysteine, i.e. an overall average of 0.23% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA. All the validity criteria were fullfilled.