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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:
weight of evidence
Study period:
8th to 15th June 2018
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
Remarks:
No deviations that influenced the quality or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Reference substance name:
Unknown 6
IUPAC Name:
Unknown 6
Constituent 2
Reference substance name:
Unknown 7
IUPAC Name:
Unknown 7
Constituent 3
Reference substance name:
Unknown 5
IUPAC Name:
Unknown 5
Constituent 4
Reference substance name:
Unknown 4
IUPAC Name:
Unknown 4
Constituent 5
Reference substance name:
Unknown 3
IUPAC Name:
Unknown 3
Constituent 6
Reference substance name:
Unknown 2
IUPAC Name:
Unknown 2
Constituent 7
Reference substance name:
Unknown 1
IUPAC Name:
Unknown 1
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 11879017 (first solubility test), 4898318 (second solubility test and main experiments)
- Expiration date of the lot/batch: August 2018 (11879017), March 2019 (4898318)
- Purity test date: Not specified
- Storage condition of test material: ≤ - 20°C

In chemico test system

Details on the study design:
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials.< The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions.

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days, all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Data Analysis
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula: PPD = (1-(Peptide Peak Area in the Replicate Injection/Mean Peptide Peak Area in Reference Control C))*100
The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly.
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration different from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

Results and discussion

Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides.
The mean depletion of both peptides was 64.83%.
Depletion of the lysine peptide: 58.47%; SD of peptide depletion: 2.23%; CV of peptide depletion: 3.81%.
Depletion of the cysteine peptide: 71.18%; SD of peptide depletion: 0.06%; CV of peptide depletion: 0.08%.
Prediction Model 1 (cysteine peptide and lysine peptide / ratio 1:10 amd 1:50): mean peptide depletion: 64.83%, reactivity category: high, prediction: sensitiser.
Prediction Model 2 (cysteine peptide / test item ratio: 1:10): 71.18%, reactivity category: moderate reactivity, prediction: sensitiser.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Parameter:
other: mean peptide depletion
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)
Parameter:
other: mean peptide depletion
Value:
71.59
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: lysine peptide depletion (%)
Value:
87.18
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
Co-elution control
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: cysteine peptide depletion (%)
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Co-elution control
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
All acceptance criteria were met.

Any other information on results incl. tables

Please refer to document attached below (OECD 442C Study 179042 Tables of Results) .

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. The test item was soluble in water. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiments were prepared using water. All test item solutions were freshly prepared immediately prior to use.

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the maximum solubility of 12.5 mg/mL solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the maximum solubility of 12.5 mg/mL solution of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed high reactivity towards both peptides. The test item is considered as “sensitiser”.
The data generated with this test item should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study Escherichia coli dehalogenase catalyst was dissolved in water, based on the results of the pre-experiments. Since no molecular weight could be derived the test item was tested to its maximum solubility which was found to be 12.5 mg/mL. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC analysis.

All test item solutions were freshly prepared immediately prior to use.

For the maximum solubility solution of 12.5 mg/mL of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged and transferred into other vials prior to the HPLC analysis.

For the maximum solubility solution of 12.5 mg/mL of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged transferred into other vials prior to the HPLC analysis. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Precipitation of the test item with both peptide peaks was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (71.59%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.83%.

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