Potassium hexafluorophosphate

Endpoint summary

Description of key information

-       in chemico skin sensitisation (DPRA, OECD 442C): no indication of sensitisation. Precipitation in cysteine peptide samples occurred.

-       in vitro skin sensitisation (KeratinoSens^TM, OECD 442D): no indication of sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-26 to 2017-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: February 04, 2015

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol n°155

Version / remarks:

July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™): The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers represents the second key event of the skin sensitisation process. This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.
- Details on study design / experimental procedure:
A cell suspension of 8.00E+04 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1.00E+04 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5 % CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5 % CO2.
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.
Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5 % CO2. Afterwards the medium was removed and replaced by 200 µL 10 % SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5 % CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
For detailed information on materials, please see section “any other information on materials and methods incl. tables”.
Data Analysis
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results, a third independent run is performed. For the parameters calculated, please see figure 1 in section “illustration (picture/graph)”.
For every concentration showing > 1.5 fold luciferase activity induction, statistical significance (p < 0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
The lowest concentration with > 1.5 fold luciferase activity induction was the value determining the EC 1.5 value. It was checked in each case whether this value was below the IC 30 value, indicating that there was less than 30 % reduction on cellular viability at the EC 1.5 determining concentration.
Prediction Model:
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5 fold increased and statistically significant (p < 0.05) compared to the negative control
- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5
- EC 1.5 value is < 1000 µM an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.
Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC 1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is < 20 % in each repetition.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.92 in experiment 1; 4.55 in experiment 2). The calculated EC1.5 was between 7 and 34 µM (16.35 µM in experiment 1; 12.29 µM in experiment 2). Thus, the results fulfil the given criteria and the positive controls are considered valid. For details please see section “any other information on results incl. tables”.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.08

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 value could be calculated. The corresponding cell viability was 95.1 %. No sign of sensitisation.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.17

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 value could be calculated. The corresponding cell viability was 99.7 %. No sign of sensitisation.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported.
DEMONSTRATION OF TECHNICAL PROFICIENCY: the measured values are well within the historical range.
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

In the first experiment, a max luciferase activity (Imax) induction of 1.08 was determined at a test item concentration of 1.95 µM. The corresponding cell viability was 95.1 %. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, a max luciferase activity (Imax) induction of 1.17 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 99.7 %. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.92 in experiment 1; 4.55 in experiment 2). The calculated EC1.5 was between 7 and 34 µM (16.35 µM in experiment 1; 12.29 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (11.1 % in experiment 1; 12.7 % in experiment 2).

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	100	100	100	0.0
Positive control	4.00	95.5	98.9	97.2	2.4
	8.00	104.9	102.4	103.7	1.8
	16.00	120.2	104.5	112.4	11.1
	32.00	119.6	112.8	116.2	4.8
	64.00	123.6	119.1	121.3	3.2
Test item	0.98	125.2	95.8	110.5	20.8
	1.95	95.1	97.4	96.3	1.6
	3.91	96.8	92.3	94.6	3.2
	7.81	102.5	99.7	101.1	2.0
	15.63	100.5	83.7	92.1	11.9
	31.25	93.8	85.0	89.4	6.2
	62.50	104.1	85.5	94.8	13.2
	125.00	92.7	98.8	95.7	4.3
	250.00	82.6	80.8	81.7	1.3
	500.00	79.7	87.8	83.7	5.8
	1000.00	72.7	71.7	72.2	0.7
	2000.00	66.7	46.1	56.4	14.5

 

Table 2: Induction of Luciferase Activity Experiment 1. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1	Concentration [µM]	Fold induction
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.0
Positive control	4.00	1.20	1.24	1.11	1.18	0.07
	8.00	1.26	1.36	1.26	1.30	0.06
	16.00	1.53	1.53	1.38	1.48	0.09
	32.00	2.81	2.22	2.12	2.38 *	0.37
	64.00	4.08	4.13	3.53	3.92 *	0.33
Test item	0.98	1.05	1.05	1.09	1.06	0.02
	1.95	0.99	1.08	1.16	1.08	0.08
	3.91	0.94	0.95	0.95	0.94	0.01
	7.81	1.09	0.98	0.98	1.02	0.07
	15.63	1.09	1.07	1.02	1.06	0.04
	31.25	1.01	1.02	0.92	0.98	0.06
	62.50	0.91	1.13	0.90	0.98	0.13
	125.00	1.05	1.09	1.01	1.05	0.04
	250.00	1.05	1.11	0.96	1.04	0.08
	500.00	1.09	1.14	0.73	0.99	0.22
	1000.00	0.91	1.00	0.88	0.93	0.06
	2000.00	0.91	1.04	0.87	0.94	0.09

 

Table 3: Induction of Luciferase Activity Experiment 2. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1	Concentration [µM]	Fold induction
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.0
Positive control	4.00	1.03	1.21	1.13	1.12	0.09
	8.00	1.28	1.38	1.30	1.32	0.05
	16.00	1.57	1.75	1.65	1.66 *	0.09
	32.00	2.43	2.71	2.42	2.52 *	0.16
	64.00	3.82	5.53	4.29	4.55 *	0.88
Test item	0.98	1.02	0.96	1.00	0.99	0.03
	1.95	0.94	1.05	1.13	1.04	0.10
	3.91	0.89	1.12	0.94	0.98	0.12
	7.81	0.93	1.11	1.47	1.17	0.27
	15.63	0.90	1.20	0.92	1.01	0.16
	31.25	0.94	1.19	0.92	1.01	0.15
	62.50	0.97	1.24	0.93	1.05	0.16
	125.00	1.05	1.16	0.88	1.03	0.14
	250.00	0.97	1.10	0.86	0.98	0.12
	500.00	0.98	1.01	0.95	0.98	0.03
	1000.00	0.95	1.04	0.80	0.93	0.12
	2000.00	0.77	0.91	0.65	0.78	0.13

 

Table 4: Induction of Luciferase Activity - Overall Induction. * = significant induction according to Student’s t-test, p < 0.05.

Overall induction	Concentration [µM]	Fold induction
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	1.00	1.00	1.00	0.00
Positive control	4.00	1.18	1.12	1.15	0.04
	8.00	1.30	1.32	1.31	0.02
	16.00	1.48	1.66	1.57 *	0.12
	32.00	2.38	2.52	2.45 *	0.10
	64.00	3.92	4.55	4.23 *	0.45
Test item	0.98	1.06	0.99	1.03	0.05
	1.95	1.08	1.04	1.06	0.02
	3.91	0.94	0.98	0.96	0.03
	7.81	1.02	1.17	1.09	0.11
	15.63	1.06	1.01	1.03	0.04
	31.25	0.98	1.01	1.00	0.02
	62.50	0.98	1.05	1.01	0.05
	125.00	1.05	1.03	1.04	0.01
	250.00	1.04	0.98	1.01	0.04
	500.00	0.99	0.98	0.98	0.01
	1000.00	0.93	0.93	0.93	0.00
	2000.00	0.94	0.78	0.86	0.11

 

Table 5: Additional Parameters; n.a. = not applicable

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5 [µM]	n.a.	n.a.	-	-
Imax	1.08	1.17	1.12	0.06
IC30 [µM]	1444.08	1067.15	1255.61	266.53
IC50 [µM]	n.a.	1848.36	1846.36	-

 

Table 6: Acceptance criteria

Criterion	Range	Experiment 1	Pass/fail	Experiment 2	Pass/fail
CV Solvent Control	< 20 %	11.1	pass	12.7	pass
No. of positive control concentration steps with significant luciferase activity induction > 1.5	≥1	2.0	Pass	3.0	Pass
EC1.5PC	7 < x < 34 µM	16.35	Pass	12.29	Pass
Induction PC at 64 µM	2 < x < 8	3.92	pass	4.55	pass

 

Table 7: Historical data

Acceptance criterion	Range	Mean	SD	n
CV Solvent Control	< 20 %	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction > 1.5	≥ 1	2.3	0.6	41
EC1.5PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2 < x < 8	3.3	1.1	41

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Executive summary:

In this guideline study according to OECD 442d (adopted: February 04, 2015), the skin sensitising potential of potassium hexafluorophosphate was assessed via its potential to induce the Keap1-Nrf2-ARE signalling pathway followed by quantification of luciferase gene expression.

Cells were incubated with potassium hexafluorophosphate for 48 h at 37 °C at the following concentrations [µM]: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 (solvent control). Afterwards, the test substance was removed, the cells were lysed and luminescence subsequently measured with a plate reader. Besides the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method.

The KeratinoSens^TM assay is considered to provide positive results if the following conditions are all met in two of two independent experimental repetitions:

- I_max is > 1.5 fold increased and statistically significant (p < 0.05) compared to the negative control

- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5

- EC 1.5 value is < 1000 µM

- there is an apparent overall dose-response for luciferase induction

In the first experiment, a max luciferase activity (I_max) induction of 1.08 was determined at a test item concentration of 1.95 µM. The corresponding cell viability was 95.1 %. In the second experiment, a max luciferase activity (I_max) induction of 1.17 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 99.7 %. Thus, in two independent experiments, no dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.92 in experiment 1; 4.55 in experiment 2). The calculated EC_1.5was between 7 and 34 µM (16.35 µM in experiment 1; 12.29 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (11.1 % in experiment 1; 12.7 % in experiment 2).

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens^TM cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser. However, the data generated with this method may be not sufficient to conclude definitely on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-07-28 to 2017-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: February 04, 2015

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)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use. 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.
Solvent as applied in the test
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). The test item was soluble in acetonitrile and was therefore chosen as the suitable vehicle. No turbidity, precipitation and phase separation were observed for the test item solutions.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The DPRA is supposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.
This test may be used for the hazard identification of sensitising chemicals in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the DPRA will be classified into UN GHS “Category 1”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”.

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 65.62 %. Thus, the positive control results are considered valid.

Key result

Run / experiment:

other: CYSTEINE PEPTIDE

Parameter:

other: % Depletion of Peptide

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Precipitation after the incubation period in the cysteine peptide samples containing also test item was observed. The obtained negative result may reflect an underestimation of the reactivity.

Key result

Run / experiment:

other: LYSINE PEPTIDE

Parameter:

other: % Depletion of Peptide

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
DEMONSTRATION OF TECHNICAL PROFICIENCY: the results of the controls are well within the historical range.
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

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation were observed for the test item solutions. All test item preparations of the main experiment were prepared using acetonitrile.

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM 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. Slight precipitation was observed for the samples of the test item (excluding the co-elution control of the test item), the reference control C_acetonitrile, and for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM 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. Phase separation (small droplets on the bottom of the vial) was observed for the samples of the positive control including co-elution control of the positive 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 precipitations or phase separation were regarded as insignificant.

Co-elution with the Peptide Peaks

No co-elution of the test item with any of the peptide peaks was observed.

Results Calibration Curve

Table 6: Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4946.3618	0.5340	4355.7368	0.5340
STD2	2473.8633	0.2670	2207.4150	0.2670
STD3	1198.9833	0.1335	1077.4733	0.1335
STD4	613.2764	0.0667	534.9317	0.0667
STD5	313.3556	0.0334	267.5728	0.0334
STD6	155.0795	0.0167	133.3614	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Based on these results, linear regression was performed and the following calibration curves were determined:

Cysteine Peptide Calibration Curve : y = 9265.42x - 5.69 ; R² = 0.9999

Lysine Peptide Calibration Curve : y = 8180.90x - 3.44 ; R² = 0.9999

Results of the Cysteine Peptide Depletion

Table 7: Depletion of the Cysteine Peptide

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	1367.6532	0.1482	70.48	70.90	0.44	0.62
	1350.3103	0.1464	70.86
	1327.2146	0.1439	71.36
Test Item	4768.4106	0.5153	0.00	0.00	0.00	n/a
	4740.9941	0.5123	0.00
	4710.5527	0.5090	0.00

n/a: not applicable

Results of the Lysine Peptide Depletion

Table 8: 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	1660.4912	0.2034	59.71	60.33	0.55	0.92
	1627.6788	0.1994	60.51
	1616.5613	0.1980	60.78
Test Item	4125.4541	0.5047	0.00	0.00	0.00	n/a
	4132.4927	0.5056	0.00
	4136.1455	0.5060	0.00

n/a: not applicable

Detailed results about the reference controls can be found in table 15.

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered. However, due to the observed precipitation after the incubation period in the cysteine peptide samples containing also test item, the obtained negative result may reflect an underestimation of the reactivity. Therefore, a conclusion on the lack of reactivity cannot be drawn with sufficient confidence and a prediction cannot be made.

Table 9: Categorization of the Test Item

Predicition 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 Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.00	Minimal reactivity	n/a	0.00	Minimal reactivity	n/a
Positive Control	65.62	High reactivity	sensitizer	70.90	Moderate reactivity	sensitizer

n/a: not applicable due to precipitation in the cysteine experiment

Acceptance Criteria

Table 10: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R^2> 0.99	0.9999	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5128	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.5007	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5007	pass
CV of the peak area of RC B	< 15 %	0.93	pass
CV of the peak area of RC C (PC)	< 15 %	0.84	pass
CV of the peak area of RC C (TI)	< 15 %	0.84	pass
mean peptide depletion of the PC	60.8 % < x < 100 %	70.90	pass
SD of peptide depletion of the PC replicates	< 14.9 %	0.44	pass
SD of peptide depletion of the TI replicates	< 14.9 %	0.00	pass

Table 11: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9999	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.4993	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.5042	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5042	pass
CV of the peak area of RC B	< 15 %	0.80	pass
CV of the peak area of RC C (PC)	< 15 %	0.32	pass
CV of the peak area of RC C (TI)	< 15 %	0.32	pass
mean peptide depletion of the PC	40.2 % < x < 69.0 %	60.33	pass
SD of peptide depletion of the PC replicates	< 11.6 %	0.55	pass
SD of peptide depletion of the TI replicates	< 11.6 %	0.00	pass

Historical data

Table 12: Historical Data Cysteine Peptide

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9993	0.0005	22
mean peptide concentration of reference A [mM]	0.4986	0.0193	22
mean peptide concentration of reference C [mM]	0.4913	0.0164	22
CV of the peak area of control B [%]	2.43	1.43	22
CV of the peak area of control C [%]	1.73	1.25	22
mean peptide depletion of the PC [%]	73.17	2.15	22
SD of peptide depletion of the PC replicates [%]	0.75	0.52	22
SD of peptide depletion of the test items [%]	1.43	1.72	79

Table 13: Historical Data Lysine Peptide

Lysine Peptide
	mean	SD	N
linearity of the calibration curve	0.9999	0.0001	19
mean peptide concentration of reference A [mM]	0.4925	0.0170	19
mean peptide concentration of reference C [mM]	0.4906	0.0181	19
CV of the peak area of control B [%]	0.33	0.02	19
CV of the peak area of control C [%]	0.70	0.83	19
mean peptide depletion of the PC [%]	57.96	5.72	20
SD of peptide depletion of the PC replicates [%]	1.69	1.53	20
SD of peptide depletion of the test items [%]	0.65	0.74	63

Table 14:   Exemplary Analysis Sequence

Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9 Run 10 Run 11	STD1 STD2 STD3 STD4 STD5 STD6 SDT7 (DB) Reference Control A, replicate 1 Reference Control A, replicate 2 Reference Control A, replicate 3
Run 12 Run 13	Co-Elution Control Positive Control Co-Elution Test Item 1
Run 14 Run 15 Run 16	Reference Control B, replicate 1 Reference Control B, replicate 2 Reference Control B, replicate 3
Run 17 Run 18 Run 19	Reference Control C, replicate 1 Positive Control, replicate 1 Test Item 1, replicate 1
Run 20 Run 21 Run 22	Reference Control C, replicate 2 Positive Control, replicate 2 Test Item 1, replicate 2
Run 23 Run 24 Run 25	Reference Control C, replicate 3 Positive Control, replicate 3 Test Item 1, replicate 3
Run 26 Run 27 Run 28	Reference Control B, replicate 4 Reference Control B, replicate 5 Reference Control B, replicate 6

Table 15: Results of the Reference Controls for the Cysteine Peptide

Cysteine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	4706.4341	4745.3830	33.7902	0.71	0.5086	0.5128	0.0036	0.71
Reference A 2	4762.8535				0.5147
Reference A 3	4766.8613				0.5151
Reference B 1	4658.0098	4603.1874	42.7989	0.93	0.5033	0.4974	0.0046	0.93
Reference B 2	4656.2378				0.5032
Reference B 3	4583.0986				0.4953
Reference B 4	4587.1167				0.4957
Reference B 5	4574.5996				0.4943
Reference B 6	4560.0620				0.4928
Reference C 1 (PC solvent)	4667.9434	4633.5290	39.1358	0.84	0.5044	0.5007	0.0042	0.84
Reference C 2 (PC solvent)	4641.6860				0.5016
Reference C 3 (PC solvent)	4590.9575				0.4961
Reference C 1 (TI solvent)	4667.9434	4633.5290	39.1358	0.84	0.5044	0.5007	0.0042	0.84
Reference C 2 (TI solvent)	4641.6860				0.5016
Reference C 3 (TI solvent)	4590.9575				0.4961

Table 16:   Results of the Reference Controls for the Lysine Peptide

Lysine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	4053.5723	4081.3554	24.1363	0.59	0.4959	0.4993	0.0030	0.59
Reference A 2	4093.3406				0.5008
Reference A 3	4097.1533				0.5012
Reference B 1	4086.7600	4140.6805	33.0556	0.80	0.5000	0.5066	0.0040	0.80
Reference B 2	4130.0015				0.5053
Reference B 3	4142.8394				0.5068
Reference B 4	4134.9473				0.5059
Reference B 5	4168.4224				0.5100
Reference B 6	4181.1128				0.5115
Reference C 1 (PC solvent)	4135.2852	4121.4758	13.3511	0.32	0.5059	0.5042	0.0016	0.32
Reference C 2 (PC solvent)	4108.6357				0.5026
Reference C 3 (PC solvent)	4120.5064				0.5041
Reference C 1 (TI solvent)	4135.2852	4121.4758	13.3511	0.32	0.5059	0.5042	0.0016	0.32
Reference C 2 (TI solvent)	4108.6357				0.5026
Reference C 3 (TI solvent)	4120.5064				0.5041

 

Interpretation of results:

other: Expert judgement: no indication of sensitisation

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the peptides. Due to precipitation in the cysteine peptide samples containing also test item prior to the HPLC analysis, the reactivity of the test item might be underestimated and a prediction cannot be made.

Executive summary:

In an in chemico skin sensitisation study performed according to OECD test guideline 442C (Direct Peptide Reactivity Assay), the reactivity of potassium hexafluorophosphate was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

A 100 mM stock solution of potassium hexafluorophosphate in acetonitrile 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.

For both 100 mM solutions of the test item no turbidity or precipitation was observed when diluted with the peptide solutions. Since the acceptance criteria for the depletion range of the positive control were fulfilled, minor observed precipitations or phase separation were regarded as insignificant. No co-elution of test item with the peptide peaks was observed.

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). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38 % (0.00 %). In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS “No Category” if the mean depletion of both peptides is below 6.38 %.

Due to the observed precipitation after the incubation period in the cysteine peptide samples containing also test item, the obtained negative result may reflect an underestimation of the reactivity. Therefore, a conclusion on the lack of reactivity cannot be drawn with sufficient confidence and a prediction cannot be made.

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 65.62 %.

In conclusion, in this study under the given conditions the test item potassium hexafluorophosphate showed minimal reactivity towards the peptides. Due to precipitation in the cysteine peptide samples containing also test item prior to the HPLC analysis, the reactivity of the test item might be underestimated and a prediction cannot be made.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitising potential of potassium hexafluorophosphate was examined in two OECD guideline studies, an in chemico (Direct Peptide Reactivity Assay (DPRA), OECD 442C) study and an in vitro approach measuring activation of the ARE-Nrf2 pathway in human keratinocytes (KeratinoSens^TM, OECD 442D).

Firstly, in the in chemico approach (Direct Peptide Reactivity Assay), the mean depletion of both peptides was≤6.38 % (0.00 %). In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS “No Category” if the mean depletion of both peptides is below 6.38 %. However, due to the observed precipitation after the incubation period in the cysteine peptide samples containing test item, the obtained negative result may reflect an underestimation of the reactivity. Therefore, a conclusion on the lack of reactivity cannot be drawn with sufficient confidence and a prediction cannot be made.

To verify the result of the DPRA study, where basically no indication of sensitisation was observed, an in vitro OECD guideline study using the KeratinoSens^TM model was conducted. Under the given conditions, potassium hexafluorophosphate did not induce the luciferase activity in the transgenic KeratinoSens^TM cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser according to this assay.

The Direct Peptide Reactivity Assay and the KeratinoSens^TM model are validated test methods for the assessment of skin sensitisation which have not been developed as stand-alone test methods, but to be used in a Weight-of-Evidence approach. When used in an AOP-based IATA, the outcome of these studies targets key events along the defined toxicity pathway and the results enable a regulatory decision. Taken together, the results obtained here in both OECD guideline studies support the classification of potassium hexafluorophosphate as a non-sensitiser.

Justification for classification or non-classification

Two OECD guideline studies (in chemico and in vitro) support the classification of potassium hexafluorophosphate as a non-sensitiser.