Potassium hexafluorophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-18 to 2015-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

26 September, 2014

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation 440/2008, Method B.40 BIS: “In Vitro Skin Corrosion: Human Skin Model Test”

Version / remarks:

30 May, 2008

GLP compliance:

yes (incl. QA statement)

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: crystalline

Details on test material:

- State of aggregation: solid
- Other: crystalline powder,
Colour: white
hygroscopic

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The EpiDermTM Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin. This in vitro method allows the identification of corrosive and non-corrosive substances and mixtures in accordance with UN GHS. It further allows a partial sub-categorisation of corrosives in a combination of optional sub-categories 1B and 1C.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied neat without addition of water to avoid potential hydrolytic degradation of the test item to hydrofluoric acid which is known to be severely corrosive.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (MatTek)
- Tissue batch number(s): 23303 Kit E
- Production date: 2015-11-18
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
- Observable damage in the tissue due to washing: not reported
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution: 5 mg/mL MTT (Lot 3V005S-34; AppliChem) in PBS (Lot 1660068; Gibco). MTT medium: MTT stock solution was diluted 1 + 4 with assay medium (Lot 111215TMB; MatTek; final concentration 1 mg/mL)
- Incubation time: 3 hours
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: according to the manufacturer: MTT QC assay, 4 hours, n=3. Acceptance criteria: OD (540-570 nm) 1.0-3.0. Result: 1.764 ± 0.045. QA statement: pass.
- Barrier function: according to the manufacturer: ET-50 assay, 100 µL 1 % Triton X-100, 4 time-points, n=3, MTT assay. Acceptance criteria: ET-50 4.77-8.72 hours. Result: 8.33 hours. QA statement: pass.
- Morphology: Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers. Results obtained with this lot conform to the requirements of the OECD TG 431 and 439.
- Contamination: sterile; no contamination.
NUMBER OF REPLICATE TISSUES: 2
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water (Lot: RNBD8066, Sigma)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration: 8 N potassium hydroxide (KOH; Lot: 10357004; Neolab)

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min experiment

Value:

ca. 95.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min experiment

Value:

ca. 62.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported.
- Direct-MTT reduction: no reduction of MTT compared to the solvent.
- Colour interference with MTT: no colouring detected.
DEMONSTRATION OF TECHNICAL PROFICIENCY: The controls confirmed the validity of the study.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 nm of two negative control tissues of the 3 min and 60 min treatment period between 0.8 and 2.8
- Acceptance criteria met for positive control: mean relative tissue viability of the two positive control tissues is ≤ 30 %
- Acceptance criteria met for variability between replicate measurements: coefficient of variation (CV) (at 20-100 % viability) between two tissues treated identically is ≤ 30 %.

Any other information on results incl. tables

Pre-Experiments

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, no determination of non-specific MTT-reduction by using killed tissues was necessary. 

The mixture of 25 mg test item per 300 µL A. dest. showed no colouring as compared to the solvent. Therefore, no determination of non-specific colour by using additional viable tissues was necessary.

Main experiments

Table 1: Resultsof 3 min experiment

* mean OD₅₇₀≥ 0.8 and ≤ 2.8

** mean relative tissue viability of the 3 min positive control ≤ 30 %

*** coefficient of variation (CV) (at 20-100 % viability) between two tissues treated identically is ≤ 30 %.

Name	Negative control		Test item		Positive control
Tissue	1	2	1	2	1	2
Absolute OD570-values	2.003	1.930	2.005	1.724	0.178	0.211
	2.025	1.919	2.017	1.747	0.176	0.218
	2.047	1.948	2.044	1.793	0.181	0.219
Mean OD570(mean of 3 aliquots)	2.025	1.932	2.022	1.755	0.178	0.216
SD	0.022	0.015	0.020	0.035	0.002	0.004
Total mean OD570 (mean of 2 replicate tissues)	1.979 *		1.888		0.197
SD of mean of 2 replicate tissues	0.066		0.189		0.027
Mean relative tissue viability [%]	100.0		95.4		10.0 **
Coefficient of variation [%] ***	3.3		10.0		13.5

 

Table 2: results of 60 min experiment

* mean OD₅₇₀≥ 0.8 and ≤ 2.8

** coefficient of variation (CV) (at 20-100 % viability) between two tissues treated identically is ≤ 30

Name	Negative control		Test item		Positive control
Tissue	1	2	1	2	1	2
Absolute OD570-values	1.885	1.991	1.405	1.017	0.149	0.124
	1.866	1.942	1.404	1.008	0.150	0.126
	1.936	2.013	1.436	1.049	0.156	0.127
Mean OD570 (mean of 3 aliquots)	1.895	1.982	1.415	1.025	0.152	0.126
SD	0.036	0.037	0.018	0.022	0.004	0.002
Total mean OD570 (mean of 2 replicate tissues)	1.939 *		1.220		0.139
SD of mean of 2 replicate tissues	0.061		0.276		0.018
Mean relative tissue viability [%]	100.0		62.9		7.1
Coefficient of variation [%] **	3.2		22.6		13.3

 

Table 3: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK (3 min experiment)	1.979	≥0.8 and ≤2.8	pass
Mean Absolute OD570 nmNK (60 min experiment)	1.939	≥0.8 and ≤2.8	pass
Mean Relative Tissue Viability [%] of PC (3 min Experiment)	10	≤ 30 %	pass
Max. CV of Viability[%]	22.6	≤ 30 %	pass

 

Table 4: historical data. Historical data were generated from 2010 to 2015.

	Mean	SD	n
Absolute OD570nm NK (3 min experiment)	1.984	0.365	33
Absolute OD570nm NK (60 min experiment)	1.959	0.331	33
Relative Tissue Viability [%] of PC (3 min Experiment)	17.6	4.65	33
Max. CV of Viability [%]	18.5	13.10	33

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Expert judgement: not corrosive

Conclusions:

In this study under the given conditions the neat test item showed no corrosive effects. The relative mean tissue viability after 3 min treatment was ≥ 50 % and after 60 min treatment ≥ 15 %. It has to be pointed out that the test item was applied neat, without addition of water.

Executive summary:

In this study according to OECD guideline 431, the potential of potassium hexafluorophosphate to induce skin corrosion was analyzed by using a three-dimensional human skin model (RhE). Neat potassium hexafluorophosphate was applied topically for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

All controls confirmed the validity of the study.

Neat potassium hexafluorophosphate showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50 % (95.4 %) after 3 min treatment and ≥ 15 % (62.9 %) after 60 min treatment.

According to this guideline study, potassium hexafluorophosphate is classified as “non-corrosive”.It has to be pointed out that the test item was applied neat without addition of water to avoid potential hydrolytic degradation to hydrofluoric acid which is known to be severely corrosive. In consequence, this study was repeated under the same conditions with the exception that the surface of the RhE was moistened with a small amount of water to check whether this would have an impact on the test result “non-corrosive”.