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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:
weight of evidence
Study period:
2019-01-14 to 2019-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
February 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
January 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In chemico test system

Details on study design:
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.

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 67.86%.

In vitro / in chemico

Results
Key result
Parameter:
other: mean peptide depletion of both peptides [%]
Value:
0.12
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS: All acceptance criteria are fulfiled
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Conc. [nM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.7360

4.8660

4.6910

0.1551

0.1593

0.1537

69.56

68.73

69.85

69.38

0.58

0.84

Test item

15.5650

15.5010

15.5250

0.5050

0.5029

0.5037

0.00

0.39

0.23

0.21

0.19

94.37

  

Table 2: Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.4950

4.6220

4.7210

0.1644

0.1690

0.1726

67.20

66.27

65.55

66.34

0.83

1.25

Test Item

13.7810

13.7280

13.6890

0.5029

0.5010

0.4996

0.00

0.00

0.10

0.03

0.06

173.21

 

Table 3: Categorization of the Test Item

Prediction Model

Prediction Model 1

Prediction model 2 8Cysteine Peptide 7 Test Item Ratio: 1:10)

Test substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

0.12

Minimal Reactivity

Negative

0.21

Minimal Reactivity

Negative

Positive Control

67.86

High Reactivity

Positive

69.38

Moderate Reactivity

Positive

 

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no-co-elution was observed, prediction model 1 based on the combination of cysteine and lysine depletion should be considered.

Applicant's summary and conclusion

Interpretation of results:
other: no peptide binding
Remarks:
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Conclusions:
The test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay according to OECD guideline 442C. The test item is considered as "non-sensitiser".
Executive summary:

In this study the test item was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 73.1 g/mol a 100 mM stock solution was prepared. 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.
All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item no turbidity or precipitation 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. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM stock solution of the test item no turbidity or precipitation 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. 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.
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.
No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C acetonitrile).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.12%). Based on the prediction model 1 the test item can be considered as non-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 67.86%.
In conclusion the test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay and the test item is considered as "non-sensitiser".