Triphenylphosphine oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 October 2018 - 15 November 2018

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:

2019

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))

Version / remarks:

04. Feb. 2015

Deviations:

yes

Remarks:

see "Part Results"

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM (European Union Reference Laboratory for alternatives to animal testing): “DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing.”

Version / remarks:

29. Jun. 2015

Deviations:

yes

Remarks:

see "Part Results"

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Triphenylphosphine oxide

EC Number:

212-338-8

EC Name:

Triphenylphosphine oxide

Cas Number:

791-28-6

Molecular formula:

C18H15OP

IUPAC Name:

(diphenylphosphoroso)benzene

Test material form:

solid

In chemico test system

Details on the study design:

Synthetic peptides:
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, are used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Instruments and Devices:
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A

Heating chamber, Centrifuge, Fridge, Freezer, Repeater pipette, pH-meter, Analytical scale, Analytical scale, Pipette 100 – 1000 µL, Glass thermometer, Ultrasonic bath, Conductometer, Carbon analyser

Chemicals:
Water for chromatography
H2O, Honeywell, HPLC grade
Demineralised water
H2O, from ion exchange cartridge. Total organic carbon (TOC) < 1 ppm
Acetonitrile for chromatography
CH3CN, ACN, Honeywell, HPLC grade
CH3CN, ACN, AppliChem, analysis grade
Trifluoroacetic acid
TFA, Merck, for spectroscopy
Hydrochloric acid
HCl, 1 M, p.a.
Ammonium hydroxide
NH3,25 %, p.a.
Ammonium acetate,
CH3COONH4, p.a, Sigma Aldrich
Disodium hydrogen phosphate,
Na2HPO4 * 2 H2O, p.a., Carl Roth

Buffers:
25 mM Phosphate buffer
1.1105 g disodium hydrogen phosphate dihydrate were dissolved in demineralised water, pH was adjusted to 7.5 with 1 M HCl (final volume 250 mL).
25 mM Ammonium acetate
481.9 mg ammonium acetate (anhydrous) were dissolved in demineralised water, pH was adjusted to 10.2 with 25 % ammonium hydroxide (final volume 250 mL).

Positive control:
Cinnamaldehyde (CAS 104-55-2, food grade ≥ 95 %),100 mM solution in acetonitrile for the cysteine peptide
2,3-Butanedione (CAS 431-03-8, > 97 %), 100 mM solution in acetonitrile for the lysine peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
0.667 mM Cys-Peptide solution was prepared by dissolving 22.5 mg of the peptide in 45 mL phosphate buffer, pH 7.5.
0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45 mL ammonium acetate buffer, pH 10.2.
0.667 mM Lys-Peptide solution was prepared by dissolving 18.1 mg of the peptide in 35 mL ammonium acetate buffer, pH 10.2. (experiment 2)
0.667 mM Lys-Peptide solution was prepared by dissolving 20.7 mg of the peptide in 40 mL ammonium acetate buffer, pH 10.2. (experiment 3)

Test item stock solution:
The test item stock solution is freshly prepared for each assay. 100 mM test item solution is prepared by dissolving 83.7 mg test item in 3 mL of the solvent acetonitrile for the Cys-Peptide and 83.7 mg test item for the Lys-peptide in experiment 1, respectively.
In experiment 2 and 3 the test item solution was prepared with 83.5 mg test item for the Lys-peptide measurement.

Peptide calibration standards:
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM peptide. Blank dilution buffer was also measured. Calibration samples were analysed before the samples containing the test item.

Test item samples:
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions described. A final volume of 1 mL per sample was prepared for each sample.

Incubation:
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 °C for 22 h and 25 min for the Cys-peptide and 22 h and 15 min for the Lys-peptide in experiment 1, respectively. In experiments 2 and 3 the incubation time was 22 h for the Lys-peptide. None of the Cys-peptide replicates were turbid after incubation in experiment 1. All three replicates for the Lys-peptide were turbid after incubation in experiment 1. They were centrifuged (10 min, 400 g) and only the clear supernatant was used for the measurement. In Experiment 2 and 3, none of the Lys-peptide replicates were turbid after incubation.

Measurements
HPLC system with UV/VIS-Detector

Evaluation of results:
Evaluation criteria of results according to the cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
0- ≤ 6.385 minimal or no reactivity negative

Evaluation criteria of results according to the cysteine 1:10 prediction model.
mean Cys peptide depletion [%] Reactivity Evaluation
> 98.24 - ≤ 100 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
0 - ≤ 13.89 minimal or no reactivity negative

Acceptance criteria
The standard calibration curve should have an r² > 0.99
The mean peptide concentration of solvent control samples of sets A and C should be 0.50 ± 0.05 mM
The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3-butanedione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
The standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion.

Results and discussion

Positive control results:

see "Any other information on results incl. tables"

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion for Cys-Peptide (%)

Value:

1.39

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion for Lys-Peptide (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 2nd experiment

Parameter:

other: mean peptide depletion for Lys-Peptide (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: 3rd experiment

Parameter:

other: mean peptide depletion for Lys-Peptide (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Key result

Parameter:

other: mean peptide depletion for both peptides (%)

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline were tested using the analysis method described. All ten proficiency chemicals showed the expected DPRA prediction and eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline.
 
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

Table 1: Calculated peptide depletion values for the Cys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	89.40	89.74	0.30
Positive control Rep. 2	89.84
Positive control Rep. 3	89.99
Test item Rep. 1	0.54	1.39	1.05
Test item Rep. 2	1.07
Test item Rep. 3	2.57

Table 2: Calculated peptide depletion values for the Lys-Peptide in experiment 1

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	44.02	46.48	2.49
Positive control Rep. 2	46.43
Positive control Rep. 3	49.00
Test item Rep. 1	0 (-0.7)*	0.00	0.00
Test item Rep. 2	0 (-0.5)*
Test item Rep. 3	0 (-1.08)*

* Note: Negative depletion values were considered as “zero” when calculating the mean.

Table 3: Calculated peptide depletion values for the Lys-Peptide in experiment 2

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	47.06	48.61	1.50
Positive control Rep. 2	48.71
Positive control Rep. 3	50.07
Test item Rep. 1	0 (-0.08)*	0.00	0.00
Test item Rep. 2	0 (-0.03)*
Test item Rep. 3	0 (-0.52)*

* Note: Negative depletion values were considered as “zero” when calculating the mean.

Table 4: Calculated peptide depletion values for the Lys-Peptide in experiment 3

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	44.72	47.16	2.49
Positive control Rep. 2	47.04
Positive control Rep. 3	49.71
Test item Rep. 1	0 (-0.71)*	0.00	0.00
Test item Rep. 2	0 (-0.46)*
Test item Rep. 3	0 (-0.93)*

Table 5: Historical Control Data for the positive control item

Parameter	Depletion [%]	Depletion [%]
Peptide	Cys- Peptide	Lys- Peptide
Mean	81.74	32.77
Standard Deviation	8.48	3.99
Range 2σ	64.82 – 98.71	24.79 - 40.74
Conducted study	89.74	46.48 and 48.61 and 47.16

The following deviations of the guideline were observed:

The phosphate buffer used for dissolution of the Cys-peptide and the ammonium acetate buffer used for dissolution of the Lys-peptide in the experiments 1-3 were 25 mM instead of 100 mM. This was considered uncritical because the positive control confirmed that the buffer strength was sufficient.

The phosphate buffer used for dissolution of the Cys-peptide in experiment 1 was prepared by dissolving disodium hydrogen phosphate dihydrate and adjusting the pH by using 1 M HCl instead of using sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate in combination.This was considered uncritical because by dissolving disodium hydrogen phosphate in water, sodium dihydrogen ions are also formed and the pH was adjusted to the correct value.

The mean percent peptide-depletion of the positive control 2,3-butanedione was marginal out of range (the value was slightly too high) in the experiments 1, 2 and 3. This was considered uncritical, because it shows, that the test system was slightly too sensitive and even under these conditions the mean percent peptide-depletion of the test item showed a value for a clearly negative DPRA-prediction.

Table 6: Mean Peptide Depletion of both peptides

	Cys-peptide depletion [%]	Lys-peptide depletion [%]	Mean peptide depletion [%]
Experiment 1	1.39	0.00	0.70
Experiment 2	-*	0.00
Experiment 3	-*	0.00

*Note: in experiment 2 and 3, Cys-peptide was not performed, because a valid result was obtained in experiment 1.

Applicant's summary and conclusion

Interpretation of results:

other: no peptide depletion

Conclusions:

The DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item possesses no or minimal skin sensitisation potential.

Executive summary:

A study according OECD TG 442C was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated at 25 °C for 22 h and 25 min for the Cys-peptide and 22 h and 15 min for the Lys-peptide, in experiment 1 respectively. In experiments 2 and 3 the incubation time was 22 h for the Lys-peptide. The peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

 

Experiment 1 was valid for the Cys-peptide. Within the measurement of the Lys-peptide the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

In experiment 2 only the measurement of Lys-peptide was performed and the mean peptide-depletion of the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

The third experiment was performed only for the Lys-peptide again and the mean peptide-depletion of the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

In experiments 1, 2 and 3 the mean peptide-depletion of the positive control 2,3-butanedione was only marginal too high, what shows that the system was a little too sensitive.

Due to the fact, that the mean peptide-depletion of the test item shows values for a clearly negative DPRA-prediction, these experiments were considered as valid. The result from experiment 1 was confirmed by the experiments 2 and 3.

 

In conclusion, the DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item possesses no or minimal skin sensitisation potential.