Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 Sep 2014 - 21 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, in-house validated procedure, pre-OECD

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Direct peptide binding assay (DPRA) performed as described in Bauch C. et al. (2011), Toxicology in Vitro 25, 1162 – 1168.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: Direct Peptide Reactivity Assay (DPRA), assessed on 13 Nov 2013 at http://www.oecd.org

Principles of method if other than guideline:

In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

EC Number:

213-551-9

EC Name:

Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

Cas Number:

976-56-7

Molecular formula:

C19H33O4P

IUPAC Name:

diethyl (3,5-di-tert-butyl-4-hydroxybenzyl)phosphonate

Details on test material:

- Physical state: solid / white

In chemico test system

Details on the study design:

synthetic peptides (Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH; Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH)

Results and discussion

Positive control results:

The peptide depletion by the positive control was 38.86%.

In vitro / in chemico

Results

Parameter:

other: mean peptide depletion in %

Value:

0

Any other information on results incl. tables

The mean C-peptide depletion, caused by the test substance was determined to be -1.34%. The mean K-peptide depletion, caused by the test substance was determined to be -1.24%. Negative depletions were considered to be “zero” for calculation of the mean peptide

depletion, which was thus calculated to be 0.00%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal

chemical reactivity in the DPRA under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

other: minimal chemical reactivity

Executive summary:

The reactivity of the test article towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was prepared at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The following results were obtained in the DPRA: The test substance was not soluble in one of the vehicles used for the assay. Thus a homogeneous suspension in acetonitrile was used for sample preparation. The test substance was soluble in acetonitrile. The samples with the test substance and the peptide stock solutions were suspensions. After 24 hours suspensions were noticed. Thus all samples were centrifuged prior to HPLC analysis. The mean C-peptide depletion, caused by the test substance was determined to be -1.34%. The mean K-peptide depletion, caused by the test substance was determined to be -1.24%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal chemical reactivity in the DPRA under the test conditions chosen.