Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1/5/18 - 18/6/18

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

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

non-animal test method preferred

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Cas Number:

2125692-22-8

Molecular formula:

C25-31H54-66NO5S

IUPAC Name:

Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Test material form:

solid

In chemico test system

Details on the study design:

DPRA is an in chemico method which quantifies the remaining concentration of cysteine or lysine-containing peptide following 24 ± 2 hours of incubation with the test chemical at a temperature of 25 ± 2.5ºC. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with gradient elution and ultraviolet (UV) detection at 220 nm. Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model which allowed the assigning of the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitisers. One or more calibration curves were generated from analyses of standard solutions of cysteine and lysine peptides analyzed concurrently with the test samples.

Results and discussion

Positive control results:

Cinnamic aldehyde (CAS #104-55-2)
The percent cysteine depletion values for the positive control sample replicates ranged from 72.2 to 73.6%. The percent lysine depletion values for the positive control sample replicates ranged from 66.2 to 67.6%. The mean percent cysteine and lysine depletion values for the respective positive control samples were 72.8 ± 0.7% (N = 3; CV = 0.987%) and 66.9 ± 0.7% (N = 3; CV = 0.99%), respectively.
The mean percent cysteine and lysine depletion values for the respective positive control samples were in the range allowed by the OECD guideline (1). Precipitate was not present in the cysteine positive control samples upon initial preparation (i.e. 0 hours) and following approximately 24 hours of incubation.

Precipitate was not present in the lysine positive control samples upon initial preparation (i.e. 0 hours) but was present following approximately 24 hours of incubation.

In vitro / in chemico

Results

Results 1

Parameter:

other: mean percent cysteine depletion

Value:

1.47

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Parameter:

other: mean percent lysine depletion

Value:

0.923

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

DPRA testing was performed on test material, using both cysteine and lysine-containing peptides. The cysteine and lysine peptide assay sequence passed all guideline acceptance criteria (1). The test material was prepared at concentrations of approximately 100 mM.

The mean percent cysteine and lysine depletion values for BVE were 1.47 ± 1.43% (N = 3; CV = 97.5%) and 0.923 ± 0.791% (N = 3; CV = 85.6%), respectively. The test substance did not co-elute with either the cysteine or lysine-containing peptides. Precipitate was present in the cysteine and lysine test substance’s assay samples upon initial preparation of the test substance solutions (i.e. 0 hours).
Precipitate present in the cysteine and lysine test substance assay samples following 24 hours of incubation. Since both the cysteine and lysine assays were valid for the test substance, the OECD cysteine 1:10/lysine 1:50 prediction model (exhibited below) was used as a reference for the assignment of a reactivity class to the test substance

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean depletion of cysteine and lysine for test material was 1.47 ± 1.43 (N=3, CV=97.5%) and 0.923 ± 0.791 (N=3; CV=85.6%), respectively. No formal DPRA prediction could be assigned due to the lack of reactivity with the presence of precipitate. The presence of precipitate could cause an underestimation of peptide depletion, therefore a negative result cannot definitely classify a compound using the cysteine 1:10/lysine 1:50 prediction model.

Executive summary:

Three replicate solutions of test material and cysteine or lysine containing peptides at a molar ratio of 1:10 and 1:50, respectively, were incubated for 24 ± 2 hours. Quantitation of the respective peptide was determined by comparison of test samples to concurrent external standards containing the respective peptide in buffer. Peptide depletion was calculated by the comparison of mean peak area of solvent matched control samples to the test substance samples. The mean depletion of cysteine and lysine for test material was 1.47 ± 1.43 (N=3, CV=97.5%) and 0.923 ± 0.791 (N=3; CV=85.6%), respectively. No formal DPRA prediction could be assigned due to the lack of reactivity with the presence of precipitate. The presence of precipitate could cause an underestimation of peptide depletion, therefore a negative result cannot definitely classify a compound using the cysteine 1:10/lysine 1:50 prediction model.