Potassium hexafluorophosphate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: February 04, 2015

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Potassium hexafluorophosphate

EC Number:

241-143-0

EC Name:

Potassium hexafluorophosphate

Cas Number:

17084-13-8

Molecular formula:

F6P.K

IUPAC Name:

potassium hexafluoro-λ⁵-phosphanuide

Test material form:

solid

Details on test material:

Aggregate State at RT: solid
Colour: white
Storage conditions: room temperature protected from light
Physical State: solid (crystalline powder)
Purity: > 99%

In vitro test system

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.

Results and discussion

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”.

In vitro / in chemico

Results

Results 1

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.

Results 2

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.