2-Naphthalenol, 1-[2-[2-ethyl-4-[2-(2-ethylphenyl)diazenyl]phenyl]diazenyl]-, ar-heptyl derivs.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This in chemico method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using high performance liquid chromatography (HPLC).

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

1

Reference substance name:

Reaction products of 2-Ethylaniline with heptyl naphthalen-2-ol

Cas Number:

1401000-07-4

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Reaction products of 2-Ethylaniline with heptyl naphthalen-2-ol

Test material form:

solid

Specific details on test material used for the study:

Batch No.: 80-47-16
Storage Conditions: Room temperature, protected from light
Expiry Date: August 2017

In chemico test system

Details on the study design:

In the present study RED 2735 was dissolved in methanol at a concentration of 2.5 mg/mL based on the results of the pre-experiments. Since no molecular weight could be derived the test item was tested to its maximum solubility. 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.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Mean Peptide Depletion [%]

Parameter:

other: Depletion of the Cysteine Peptide

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: Mean Peptide Depletion [%]

Parameter:

other: Depletion of the Lysine Peptide

Value:

3.36

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Run / experiment:

other: Mean Peptide Depletion [%]

Parameter:

other: Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50

Value:

1.68

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the peptides. Since the test item could not be tested with a 100 mM stock solution, the obtained negative results might not reflect the true reactivity and a prediction cannot be made.
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 RED 2735 was dissolved in methanol at a concentration of 2.5mg/mL based on the results of the pre-experiments. Since no molecular weight could be derived the test item was tested to its maximum solubility. 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.

For the 2.5 mg/mL stock solution of the test item no precipitation, turbidity or phase separation was observed upon mixing of the test item and the cysteine peptide. 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 positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 2.5 mg/mL stock solution of the test item precipitation was observed upon mixing of the test item and the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected again for precipitation, turbidity or phase separation. Precipitation of the test item was not detected anymore. Turbidity was observed for the samples of the positive control including the co-elution control of the positive 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 precipitation and phase separation were regarded as insignificant.

As no co-elution of test item with the peptide peaks was observed and as the observed precipitation upon mixing of the test item and the lysine peptide solution was not detected anymore after the 24 h incubation period, 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).

The 2.5 mg/mL stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38% (1.68%). The test item would be categorized as non-sensitiser. However, since the test item was not tested with a 100 mM stock solution, the prediction might be not applicable.

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 64.30%.