Bis(2-ethylhexyl) phosphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Oct 2017 - 18 Jan 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:

2018

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:

February 04, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayer, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phosphonate

EC Number:

222-904-6

EC Name:

Bis(2-ethylhexyl) phosphonate

Cas Number:

3658-48-8

Molecular formula:

C16H35O3P

IUPAC Name:

bis(2-ethylhexyl) phosphonate

Test material form:

liquid

In chemico test system

Details on the study design:

- Details of the test procedure used: The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. If a precipitate or phase separation was observed in prior and after Incubation, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial.
A standard calibration curve was generated before the HPLC analysis of samples. Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

- Peptides used: cysteine peptide: Ac-RFAACAA; lysine peptide: Ac-RFAAKAA.

- Doses of test chemical and control substances used: test material: 100 mM stock solution of test substance; negative control: acetonitrile (undiluted); positive control: 100 mM stock solution of test substance.
The preparation of 1:10 Cysteine Peptide Run: 50 µL Test Item Stock Solution (100 mM) +200 µL acetonitrile +750 µL Peptide Stock Solution (0.667 mM)
The preparation of 1:50 Cysteine Peptide Run: 250 µL Test Item Stock Solution (100 mM) + 750 µL Peptide Stock Solution (0.667 mM)

- Duration and temperature of exposure period: 24 ± 2 h at 25 ± 2.5 °C.

- Co-elution Control used: the same solution preparation but use 750 µL buffer instead of Peptides, to check if test material or positive control co-elution with the Peptide Peaks.

- Number of replicates used per test chemical and controls: three replicates per treatment.

- Acceptance Criteria
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

- Evaluation criteria
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
By using the prediction model 1 (cysteine 1:10 model), the threshold of 13.89% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

Results and discussion

Positive control results:

The acceptance criteria for the depletion range of the positive control were fulfilled.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cystein

Parameter:

other: mean peptide depletion [%]

Value:

0.4

Vehicle controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: non-sensitiser

Results 2

Key result

Run / experiment:

other: lysine

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: non-sensitiser

Other effects / acceptance of results:

- The substance was dissolved in acetonitrile.
- For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution and with the lysine peptide solution.
- No co-elution of test item with the peptide peaks was observed.
- Precipitation and phase separation were observed for the samples of the positive control. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and phase separation were regarded as insignificant.

Any other information on results incl. tables

Table 1: Depletion of the CysteinePeptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1320.4471	0.1577	70.95
	1315.4908	0.1571	71.06	71.10	0.18	0.25
	1304.7782	0.1559	71.29
Test Item	4537.9878	0.5364	0.15
	4580.9971	0.5414	0.00	0.40	0.56	141.03
	4497.4966	0.5316	1.05

Table 2: Depletion of the LysinePeptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1690.0214	0.2164	56.37
	1699.1774	0.2175	56.14	56.51	0.47	0.83
	1664.2920	0.2131	57.04
Test Item	3974.3203	0.5085	0.00
	3991.4160	0.5107	0.00	0.00	0.00	--
	3932.5691	0.5031	0.00

Table 3:Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Petide Depletion [%]	Reactivity Category	Prediction
Test Item	0.20	Minimal Reactivity	no sensitiser	0.40	Minimal Reactivity	no sensitiser
Positive Control	63.81	High Reactivity	sensitiser	71.10	Moderate Reativity	sensitiser

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item can be considered as “non-sensitiser”.