1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-05-14 to 2018-05-18

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:

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:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

EC Number:

944-870-8

Molecular formula:

C18H14F20O9S3

IUPAC Name:

1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Preparation of the Test Item:
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.159 was used to correct for the purity of the test item.

Controls:
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.

Positive Control:
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution Control:
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration
curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Peptides:
19.55 mg cysteine peptide (JPT Peptide Technologies GmbH; > 95%) with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (37.19 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
18.08 mg lysine peptide (JPT Peptide Technologies GmbH; > 95%) with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (34.34 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose Groups:
Reference Control C (solvent control)
Test Item :100 mM stock solution
Positive Control: 100 mM stock solution

Pre-Experiments:
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvents suitable for the test:
- acetonitrile
- dist. water
- dist. water : acetonitrile 1:1 (v/v)
The test item was not soluble in either acetonitrile or dist. water. The test item was completely soluble in dist. water : acetonitrile 1:1 (v/v) , therefore, dist. water : acetonitrile 1:1 (v/v) was chosen as suitable vehicle for the main experiments.

Experimental Procedure
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. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using HPLC.

Preparation of the HPLC Standard Calibration Curve:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer ( v / v ) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions.

HPLC Preparation and Analysis:
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.

Data Analysis:
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated. The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was
checked for consistency between reference control and test item samples. If this ratio was not consistent a co-elution was assumed and the evaluation would be adjusted accordingly. Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value.

Prediction Model:
The test item is considered positive, i.e. to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), see tabe 3) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin, if the mean depletion of both peptides is below 6.38%. In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model). A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test
chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. In situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

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.

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.88%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

3.96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

77.88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depleation of both peptides [%]

Value:

40.92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Pre-Experiments:
Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water : acetonitrile 1:1 (v/v). No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water : acetonitrile 1:1 (v/v). All test item solutions were freshly prepared immediately prior to use.

Co-elution with the Peptide Peaks:
No co-elution of the test item with any of the peptide peaks was observed.

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: Results

Cysteine and Lysine Values of the Calibration Curve

Sample	Cystein Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	15.8430	0.5340	16.1480	0.5340
STD2	8.0650	0.2670	8.0680	0.2670
STD3	4.0830	0.1335	4.0960	0.1335
STD4	2.0190	0.0667	2.0510	0.0667
STD5	0.9700	0.0334	1.0420	0.0334
STD6	0.4700	0.0167	0.5180	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Table 2: Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.4450	0.1486	70.62	70.09	0.62	0.88
	4.5010	0.1505	70.25
	4.6270	0.1547	69.42
Test Item	15.5030	0.5203	2.73	3.96	1.09	27.42
	15.2440	0.5116	4.35
	15.1750	0.5093	4.79

Table 3: Depletion of the Lysine Peptide

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	5.6780	0.1872	62.87	61.66	1.15	1.86
	5.8860	0.1941	61.51
	6.0270	0.1988	60.59
Test Item	3.4580	0.1137	77.20	77.88	0.64	0.82
	3.3400	0.1098	77.98
	3.2660	0.1073	78.46

Table 4: Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item 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 Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	40.92	Moderate Reactivity	sensitiser		--*	--*	--*
Positive Control	65.88	High Reactivity	sensitiser		70.09	Moderate Reactivity	sensitiser

* = not applicable

Applicant's summary and conclusion

Interpretation of results:

other: peptide depletion

Conclusions:

In this study under the given conditions the test item showed moderate reactivity towards both peptides. The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

A study according OECD TG 442 C was conducted for detection of the sensitising potential of the test item by quantifying its reactivity towards synthetic peptides containing either lysine or cysteine.

The test item was dissolved in dist. water : acetonitrile 1:1 (v/v), based on the results of the pre-experiments. Considering the molecular weight of 872.45 g/mol of the test item, a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC. All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (excluding the co-elution control). Samples were centrifuged prior to the HPLC analysis. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the cysteine or lysine peptide peak was observed. A possible sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C dist. Water : Acetonitrile 1:1 (v/v)).

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptides. The mean depletion of both peptides was 40.92% and, thus, exceeded the threshold of 6.38%. Even though a precipitate was observed in the cysteine experiment a positive result can still be used. Based on the cysteine 1:10 / lysine 1:50 prediction model the test item can be considered as a skin sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.88%.

In this study under the given conditions the test item showed moderate reactivity towards both peptides. The test item is considered as skin sensitiser in this in vitro assay. However, the data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).