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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
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:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
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:
2,2'-dithiobisethanol
EC Number:
217-576-6
EC Name:
2,2'-dithiobisethanol
Cas Number:
1892-29-1
Molecular formula:
C4H10O2S2
IUPAC Name:
2-[(2-hydroxyethyl)disulfanyl]ethan-1-ol
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Based on a solubility test, the test item was dissolved in acetonitrile (ACN).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: di-2-hydroxyethyl disulfide stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assays 26.31 mg of di-2-hydroxyethyl disulfide was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1706 μL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preliminary purification step (if any): No correction for the purity/composition of the test item was performed.
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

In chemico test system

Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
- Preparation of Solutions for Cysteine Reactivity Assay:
*Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by two times 5 minutes sonication.
*SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
*SPCC Calibration Curve: A SPCC calibration curve was prepared as described in table 1.
Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in table 2.

- Preparation of Solutions for Lysine Reactivity Assay:
*Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
* SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
* SPCL Calibration Curve: A SPCL peptide calibration curve was prepared as described in table 3.
* Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive
control samples (PC) were prepared as described in table 4.

-Sample Incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24±2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

-HPLC-PDA Analysis: SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed. All samples were analyzed according to the HPLC-PDA method

- ACCEPTABILITY CRITERIA:
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine
Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other:
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: The percent SPCC depletion was calculated versus the mean SPCC peak area of reference controls C. The mean Percent SPCC Depletion for di-2-hydroxyethyl disulfide was 100.0% ± 0.0%.
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other:
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: The percent SPCC depletion was calculated versus the mean SPCC peak area of reference controls C. The mean Percent SPCC Depletion for di-2-hydroxyethyl disulfide was 2.3% ± 3.9%.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Any other information on results incl. tables

- Solubility assessment of di-2-hydroxyethyl disulfide: At a concentration of 100 mM, di-2-hydroxyethyl disulfide was soluble in ACN and therefore this solvent was used to dissolve di-2 -hydroxyethyl disulfide in this DPRA study.

- Cysteine Reactivity Assay for di-2-hydroxyethyl disulfide: Preparation of a 100 mM di-2-hydroxyethyl disulfide stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate was observed in any of the samples. In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 207985/A-cys samples, the mean SPCC A220/A258 area ratio was 24.05. This was outside the 15.93-19.47 range. However, since the test item displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm. Overall, it can be concluded that the test item did not co-elute with SPCC. The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for di-2 -hydroxyethyl disulfide was 100.0% ± 0.0%.

- Results Lysine Reactivity Assay for di-2-hydroxyethyl disulfide: Preparation of a 100 mM di-2-hydroxyethyl disulfide stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the CC as well as the test item samples were visually inspected. No precipitate was observed in any of the samples. In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 207985/A-lys samples, the mean SPCL A220/A258 area ratio was 13.49. Since this was within the 12.19-14.90 range, this again indicated that there was no co-elution of the test item with SPCL. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for di-2-hydroxyethyl disulfide was 2.3% ± 3.9%.

Table 1: SPCC and SPCL Depletion and Reactivity Classification for di-2-hydroxyethyl disulfide

Test item 

SPCC depletion 

SPCL depletion

Mean of

SPCC and

SPCL

depletion

Reactivity class

 

Mean

± SD

Mean

± SD

 

Cysteine 1:10 / Lysine 1:50 prediction model

Di-2-

hydroxyethyl disulfide

100.0%

±0.0%

2.3%

±3.9%

51.1%

High reactivity

Applicant's summary and conclusion

Interpretation of results:
other: indicative of skin sensitisation potential
Conclusions:
The DPRA test for di-2-hydroxyethyl disulfide concluded the test substance to be classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The DPRA test was concluded to be valid. The study was conducted according to appropriate OECD Test Guideline and in compliance with GLP.

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