Tris(2-methylphenyl)phosphine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2017 to 13 October 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

As per the updated Annex VII 8.3 requirements

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-methylphenyl)phosphine

EC Number:

228-193-9

EC Name:

Tris(2-methylphenyl)phosphine

Cas Number:

6163-58-2

Molecular formula:

C21H21P

IUPAC Name:

tris(2-methylphenyl)phosphane

Test material form:

solid: particulate/powder

Details on test material:

Lot number: 2000850599
Analysis date: 19 August 2016

Specific details on test material used for the study:

Chemical name: Tris(2-methylphenyl)phosphine
CAS Number 6163-58-2
Molecular formula C21H21P
Structure codes: SMILES: C1=CC(=C(C=C1)C)P(C2=C(C=CC=C2)C)C3=C(C=CC=C3)C
Appearance: Off-white crystalline powder (determined by Charles River Den Bosch)

In vitro test system

Details on the study design:

Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. In addition, a RCcysCacetone:ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCacetone:ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL acetone:ACN (1:1, v/v)

Synthetic Peptide Containing Lysine (SPCL) Stock Solution
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. In addition, a RClysCacetone:ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCacetone:ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL acetone:ACN (1:1, v/v).

Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in an incubator at 25±2.5°C. After incubation, the samples were transferred to the autosampler. The incubation time between placement of the samples in the incubator and analysis of the first RCcysB- or RClysBsample was 24.5 hours for cysteine and 25.5 hours for lysine. The time between the first
RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.

Results and discussion

Positive control results:

Positive Control Cinnamic Aldehyde

In vitro / in chemico

Results

Results 1

Parameter:

other: Cystine Reactivity Assay

Remarks:

16.1% SPCC depletion

Value:

16.1

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Parameter:

other: lysine reactivity assay

Remarks:

0.4% SPCL depletion

Value:

0.4

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Parameter:

other: mean of the synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) depletion

Value:

8.2

Positive controls validity:

valid

Any other information on results incl. tables

In the cysteine reactivity assay the test item showed 0.1 ± 0.2% SPCC depletion while in the lysine reactivity assay the test item showed 0.8 ± 1.2% SPCL depletion. The mean of the synthetic peptides containing either cysteine(SPCC) or lysine (SPCL) depletion was 0.5% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class”when using the Cysteine 1:10 / Lysine 1:50 prediction model.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion. In the cysteine reactivity assay the test item showed 16.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 8.2% and as a result the test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Tri-o-tolylphosphine was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since
precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the peptide depletion may be underestimated.

Executive summary:

The objective of this study was to determine the reactivity of Tri-o-tolylphosphine towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent

Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guideline. Acetone:acetonitrile (ACN) (1:1, v/v) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cacetone:ACN, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 16.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 8.2% and as a result the test item was considered to be positive in the DPRA

and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. Tri-o-tolylphosphine was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the

peptides. Consequently, the peptide depletion may be underestimated