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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
Study period:
7th June 2018 - 19th July 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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.
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 an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch No.: 0400
Purity: 96%
Appearance: Orange solid
Storage conditions: Room temperature

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%.

In vitro / in chemico

Results
Key result
Parameter:
other: Mean peptide depletion (%)
Run / experiment:
Test item
Value:
2.41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Reference controls
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes: The mean depletion of both peptides was 64.83%.

Any other information on results incl. tables

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water : acetonitrile 1:1 (v/v). No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water : acetonitrile 1:1 (v/v). 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 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. Phase separation was observed for the samples of the positive control including the co-elution control. No precipitation, turbidity or phase separation was observed for the samples of the test item. 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

Co-elution with the Peptide Peaks

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

Depletion of the cysteine peptide depletion:

Cysteine 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

4.7290

4.7400

4.7210

0.1451

0.1454

0.1449

71.19

71.12

71.24

 

71.18

 

0.06

 

0.08

Test item

16.1620

16.0220

15.7820

0.4951

0.4908

0.4835

2.31

3.15

4.60

 

3.35

 

1.16

 

34.65

Depletion of the Lysine peptide

Cysteine 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

6.2940

5.9870

5.6530

0.2177

0.2071

0.1955

56.28

58.41

60.73

 

58.47

 

2.23

 

3.81

Test item

14.2650

14.3900

14.3550

0.4938

0.4981

0.4969

1.95

1.10

1.34

 

1.46

 

0.44

 

30.32

Categorization of the test item:

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 peptide depletion should be considered.

Prediction model

Prediction model 1 (Cysteine peptide and lysine peptide/ratio 1:10 and 1:50)

Prediction model 2 (cysteine peptide/test item ratio: 1: 10)

Test substance

Mean peptide depletion [%]

Reactivity category

Prediction

Mean peptide depletion [%]

Reactivity category

Prediction

Test item

2.41

Minimal reactivity

No sensitiser

3.35

 

Minimal reactivity

No sensitiser

Positive control

64.83

High reactivity

Sensitiser

71.18

Moderate reactivity

sensitiser

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given conditions of the study, the test item showed minimal reactivity towards both peptides. Therefore, the test item, balsalazide acid, might be considered as a non-sensitiser.