4-pyridylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

This deviation did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.

Test material

Test material information

Constituent 1

Reference substance name:

4-pyridylamine

EC Number:

207-987-9

EC Name:

4-pyridylamine

Cas Number:

504-24-5

Molecular formula:

C5H6N2

IUPAC Name:

nickel

Test material form:

solid: particulate/powder

In chemico test system

Details on the study design:

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.

Results and discussion

Positive control results:

Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test. Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides. 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 68.75%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

other: Mean Peptide Depletion [%] of the Cysteine Peptide

Value:

0.17

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

mean

Parameter:

other: Mean Peptide Depletion [%] of the Lysine Peptide Depletion

Value:

0.04

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item 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	0.10	Minimal Reactivity	no sensitiser	0.17	Minimal Reactivity	no sensitiser
Positive Control	68.75	High Reactivity	sensitiser	74.85	Moderate Reactivity	sensitiser

Th ein chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers. In the present study 4-Aminopyridine was dissolved in acetonitrile, based on the results of the pre-experiments.Based on a molecularweight of 94.11 g/mol a 100 mM stock solutionwas 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. For the 100 mM stock solutionof 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. No precipitation, turbidity or phase separation was observed for any of the samples. For the 100 mM 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 any of the samples. No co-elution of 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% (0.10%). 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 68.75%. The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived fromin vitro assays addressing other key events of the skin sensitisation AOP.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser. 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.

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 4-Aminopyridine was dissolved in acetonitrile, based on the results of the pre-experiments.Based on a molecularweight of 94.11 g/mol a 100 mM stock solutionwas prepared. The test item solutions were testedby 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. For the 100 mM stock solutionof 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. No precipitation, turbidity or phase separation was observed for any of the samples. For the 100 mM 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 any of the samples. No co-elution of 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% (0.10%). 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 68.75%.