Ethyltriphenylphosphonium acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2019 - 14 March 2019

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:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylphosphonium acetate

EC Number:

252-743-7

EC Name:

Ethyltriphenylphosphonium acetate

Cas Number:

35835-94-0

Molecular formula:

C20H20P.C2H3O2

IUPAC Name:

ethyltriphenylphosphanium acetate

Test material form:

solid

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing Cysteine: Ac-RFAACAA-OH
Synthetic peptide containing Lysine: Ac-RFAAKAA-OH
Positive Control: Cinnamic Aldehyde

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solutions
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of ETPPAAc was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and ETPPAAc stock solutions were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM ETPPAAc. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and ETPPAAc stock solution were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 mM ETPPAAc. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the ETPPAAc and positive control samples in the HPLC vials was documented following preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of ETPPAAc and the associated positive controls was quantified by HPLC using UV detection.

The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100))/(Mean Peptide peak area of reference control samples B)

Results and discussion

Positive control results:

72.4% Cysteine depletion
51.8% Lysine depletion

In vitro / in chemico

Results

Results 1

Run / experiment:

run/experiment 1

Parameter:

cysteine depletion

Value:

-2.41 %

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

72.4% depletion

Results 2

Run / experiment:

run/experiment 1

Parameter:

lysine depletion

Value:

-0.514 %

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

51.8% depletion

Other effects / acceptance of results:

Solubility of ETPPAAc was achieved at a nominal concentration of 100 mM in acetonitrile.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

The DPRA prediction and the reactivity of the test item based on the overall mean and the individual depletion valuesin the Cysteine peptide and the Lysine peptide are presented below. All analytical acceptance criteria for each peptide run were met:

 

All analytical acceptance criteria for each peptide run were met:	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance	Cysteine	r2>0.99	60.8-100(SD<14.9%)	0.45-0.55mM(CV <15%)	SD<14.9%
criteria	Lysine	r2>0.99	40.2-69.0 (SD<11.6%)	0.45-0.55mM (CV <15%)	SD<11.6%
Achieved	Cysteine	r2>0.999	72.4(SD, 0.58%, n=3)	B: 0.504mM (CV0.64%, n=6)	SD0.48%(n=3)
results	Lysine	r2>0.999	51.8(SD, 0.60%, n=3)	B: 0.501mM (CV1.63%, n=6)	SD0.22%(n=3)

 

 

The depletion of peptide in the presence of the test item was:

Peptide	Reference Control	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by ETPPAAc (%)
Cysteine	Control B: 921660 (n=6)	941360 (n=3)	-2.41 (n=3)
Lysine	Control B: 783900 (n=6)	787930 (n=3)	-0.514 (n=3)

Applying the following reactivity prediction depletion model (below), reactivity of ETPPAAc is classed as “no or minimal” and the DPRA prediction is therefore negative and is therefore predicted not to be a potential skin sensitizer. 

 

Mean of cysteine and lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

There was no co-elution peaks in either the Cysteine or Lysine assay.   

 

 

 

Applicant's summary and conclusion

Interpretation of results:

other: expert judgement

Remarks:

negative for the first key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

negative for the first key event of the skin sensitisation Adverse Outcome Pathway

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD document TG 442C) was to assess the reactivity and sensitizing potential of ETPPAAc.

Solutions of the test item were successfully analyzed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides. There was no reactivity of both peptides in the presence of the test item. With no peptide depletion, the reactivity of the test item is classified as “no or minimal” and hence the DPRA prediction is negative. ETPPAAc is likely to be a non-skin sensitizer based as based on this assay.