Potassium hexafluorophosphate

Endpoint summary

Description of key information

-       in vitro skin corrosion: no corrosive effects (two studies, application neat and moistened)

-       in vitro skin irritation: no irritant effect

 

-       in vitro eye irritation (BCOP): mean in vitro irritation score: 9.49. No prediction can be made. Classified voluntarily as mildly irritating to eyes (UN GHS Category 2B).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 September, 2014

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 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

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

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 %.

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

 

 

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”.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 Sept. 2014

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)

Specific details on test material used for the study:

- Solid, white, crystalline powder

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:

other: The test item was moistened with 25 µL water to ensure good contact with the skin model.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (MatTek)
- Tissue batch number(s): 25806 Kit C/F
- Production date: 2017-04-19
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 (Sigma; Lot MKBR6576V) in PBS (MatTek; Lot No.: 101816ZSA). MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (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: 1.581 ± 0.238. 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: 5.58 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
VEHICLE
- Amount(s) applied (volume or weight with unit): the test item was not dissolved but moistened with 25 µL water to ensure good contact with the skin disc.
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water (Lot: RNBF7110, Sigma)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration: 8 N potassium hydroxide (KOH; CAS No.: 1310-58-3; Lot: 10357-004, NeoLab)

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min experiment

Value:

ca. 98.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min experiment

Value:

ca. 90.4

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 %.

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.

The mixture of 25 mg test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent.

Main experiments

Table 2: Results of 3 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	1.705	1.696	1.649	1.658	0.192	0.196
	1.642	1.734	1.699	1.683	0.187	0.190
	1.733	1.754	1.709	1.699	0.196	0.209
OD570- Blank Corrected	1.657	1.649	1.602	1.610	0.144	0.149
	1.595	1.687	1.651	1.635	0.140	0.143
	1.686	1.707	1.662	1.651	0.149	0.161
Mean OD570(mean of 3 aliquots)	1.646	1.681	1.638	1.632	0.144	0.151
SD	0.047	0.037	0.039	0.032	0.026	0.027
Total mean OD570of 2 replicate tissues (blank corrected)	1.663*		1.635		0.148
SD OD570of 2 replicate tissues	0.025		0.004		0.005
Mean relative tissue viability [%]	100.0		98.3		8.9
Coefficient of variation [%] ***	1.5		0.3		3.1

 

Table 3: results of 60 min experiment

* mean OD₅₇₀≥ 0.8 and ≤ 2.8

** mean relative tissue viability of the 60 min positive control < 15 %

*** 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	1.724	1.742	1.644	1.507	0.119	0.163
	1.708	1.761	1.647	1.515	0.129	0.178
	1.751	1.766	1.629	1.536	0.118	0.162
OD570- Blank Corrected	1.667	1.694	1.597	1.460	0.072	0.115
	1.661	1.714	1.600	1.468	0.082	0.131
	1.704	1.719	1.581	1.489	0.071	0.114
Mean OD570(mean of 3 aliquots)	1.680	1.709	1.592	1.472	0.075	0.120
SD	0.022	0.028	0.027	0.029	0.027	0.027
Total mean OD570of 2 replicate tissues (blank corrected)	1.695*		1.532		0.097
SD OD570of 2 replicate tissues	0.020		0.085		0.032
Mean relative tissue viability [%]	100.0		90.4		5.7**
Coefficient of variation [%] ***	1.2		5.6		32.8

 

Table 4: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK (3 min experiment)	1.663	≥0.8	pass
Mean Absolute OD570 nmNK (60 min experiment)	1.695	≥0.8	pass
Mean Relative Tissue Viability [%] of PC (60 min Experiment)	5.7	<15 %	pass
CV[%] (in the range of 20-100 % viability)	0.3-5.6	≤30 %	pass

 

Table 5: historical data. Historical data were generated from 2015 to 2016.

	Mean	SD	n
OD570nm NK (3 min experiment)	1.934	0.413	6
OD570nm NK (60 min experiment)	1.905	0.308	6
Relative Tissue Viability [%] of PC (60 min Experiment)	6.4	1.18	6
CV[%] (in the range of 20-100 % viability)	9.0	8.1	23

 

 

Interpretation of results:

other: Expert judgement: not corrosive.

Conclusions:

In this study under the given conditions the moistened test item showed no corrosive effects. The relative mean tissue viability after 3 min treatment was ≥ 50 % and after 60 min treatment ≥ 15 %.

Executive summary:

In this study according to OECD guideline 431, the potential of potassium hexafluorophosphate to induce skin corrosion was analysed by using a three-dimensional human skin model (RhE). Potassium hexafluorophosphate was moistened with a small amount of water and then 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.

In this setup, potassium hexafluorophosphate showed no corrosive effects even if it is moistened with water. The mean relative tissue viability (% negative control) was ≥ 50 % (98.3 %) after 3 min treatment and ≥ 15 % (90.4 %) after 60 min treatment.

According to this guideline study, potassium hexafluorophosphate is classified as “non-corrosive”.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-09 to 2017-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 modified in vitro EpiDerm™ Skin Irritation Test (EPI-200-SIT) is accepted as a reliable and relevant stand-alone replacement test for in vivo skin irritation testing. This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis. This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

other: 25 µL of sterile DPBS was applied to the epidermal surface in order to improve the contact between the powder and the epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (MatTek)
- Tissue batch number(s): 25835
- Production date: 2017-08-09
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation: 37 ± 1°C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: not reported
- Modifications to validated SOP: not reported
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution: 5 mg/mL MTT (Sigma; Lot MKBZ5197V) in PBS (Gibco; Lot No.: 1866243). MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 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: 1.474 ± 0.093. 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: 5.93 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: 3
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

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 (39 mg/cm2)
- Concentration: neat application, but 25 µL of sterile DPBS (Dulbecco’s phosphate buffered saline) was applied to the epidermal surface in order to improve the contact between the powder and the epidermis.
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration: 5% SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main experiment

Value:

ca. 82.7

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 detectable by unaided eye-assessment.
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 the three negative control tissues is ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: mean relative tissue viability of the three positive control tissues is ≤ 20 %
- Acceptance criteria met for variability between replicate measurements: standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18 %.

Pre-Experiments

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0 %.

The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0 %.

Main experiments

Table 1: Results.

* Blank-corrected mean OD570 nm of the negative control corresponds to 100 % absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is ≤ 20 %.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18 %

Name	NK			PC			TM
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.635	1.509	1.498	0.106	0.096	0.102	1.219	1.234	1.401
	1.595	1.511	1.510	0.110	0.098	0.102	1.219	1.233	1.399
OD570(blank-corrected)	1.592	1.466	1.455	0.062	0.053	0.059	1.176	1.191	1.358
	1.552	1.468	1.467	0.066	0.055	0.059	1.176	1.189	1.356
mean OD570of the duplicates (blank-corrected)	1.572	1.467	1.461	0.064	0.054	0.059	1.176	1.190	1.357
total mean OD570of 3 replicate tissues (blank-corrected)	1.500*			0.059			1.241
SD OD570	0.062			0.005			0.101
relative tissue viability [%]	104.8	97.8	97.4	4.3	3.6	3.9	78.4	79.3	90.5
mean relative tissue viability [%]	100.0			3.9**			82.7
SD tissue viability [%]***	4.2			0.4			6.7
CV [% viabilities]	4.2			8.9			8.1

 Table 2: quality criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.500	0.8 ≤ NK ≤2.8	pass
Relative Viability [%] PC	3.9	≤ 20%	pass
SD Viability[%]	0.4 -6.7	≤ 18%	pass

 Table 3: historical data. Historical data were generated from 2009 to 2016.

	Mean OD570±30nmNK	Mean Relative Viability [%] PC	SD Viability [%]
Mean	1.831	3.9	4.4
SD	0.357	1.9	5.1
n	27	27	88

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no irritant effects in a human epidermis model. The mean relative tissue viability (% negative control) was > 50 % (82.7 %) after 60 min treatment and 42 h post-incubation.

Executive summary:

In this study according to OECD guideline 439, the potential of potassium hexafluorophosphate to induce skin irritation was analysed by using a three-dimensional human epidermis model (RhE). The test substance was applied topically for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

All controls confirmed the validity of the study. Potassium hexafluorophosphate showed no irritant effects. The mean relative tissue viability (% negative control) was> 50 % (82.7 %) after 60 min treatment and 42 h post-incubation.

Hence, potassium hexafluorophosphate is considered “non-irritant” in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue: on the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: penicillin/streptomycin in HBSS during transport

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was administered directly and moistened with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 17185401, expiry date: 04/2020).

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 mg

Duration of treatment / exposure:

4 hours ± 5 minutes at 32 ± 1 °C.

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

90 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: eyes were carefully examined for defects and any defective eyes were discarded.
QUALITY CHECK OF THE ISOLATED CORNEAS: corneas have been visually examined for defects and any defective cornea had been discarded.
NUMBER OF REPLICATES: three
NEGATIVE CONTROL USED: yes, 750 µL physiological saline
POSITIVE CONTROL USED: yes, 750 µL imidazole 20% in physiological saline
APPLICATION DOSE AND EXPOSURE TIME: Dose: 750 mg. Exposure time: 4 hours ± 5 minutes at 32 ± 1 °C.
TREATMENT METHOD: control substance: closed chamber. Test item: open chamber
REMOVAL OF TEST SUBSTANCE: epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
POST-EXPOSURE INCUBATION: 90 minutes at 32 ± 1 °C.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: illuminance measurement.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490); Jenway 6405 UV/VIS.
- Others: each cornea was observed visually and pertinent observations were recorded.
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: according to guideline.
For further details, see section “any other information on materials and methods incl. tables”.

Irritation parameter:

in vitro irritation score

Value:

ca. 9.49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: for detailed information, see tables 4 and 5 in section “any other information on results incl. tables”.

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay.

The test item was administered directly and moistened with physiological saline 0.9 % NaCl.

All 3 corneas treated with the test substance showed slight opacity of the tissue.

The following mean in vitro irritation score was calculated: 9.49

No prediction can be made regarding the classification of the test substance according to the evaluation criteria.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

For detailed data see tables 1-3 below.

INDIVIDUAL DATA

Table 1: Opacity. MV= mean value.

Cornea Nr. 1	Test item	Initial opacity	Final opacity	Change of opacity value	Corrected opacity value
1	Negative control	3.11	3.34	0.23
2		2.65	4.39	1.74
3		3.38	4.18	0.80
MV		3.05	3.97	0.92
4	Positive control	5.01	127.80	122.78	121.86
5		5.96	123.14	117.17	116.25
6		5.10	107.75	102.65	101.73
MV		5.36	119.56	114.20	113.28
7	Test item	1.94	12.13	10.19	9.27
8		2.05	8.24	6.18	5.26
9		1.94	10.91	8.97	8.04
MV		1.98	10.43	8.45	7.52

Table 2: permeability. MV= mean value.

Cornea Nr.	Test item	OD490	Corrected OD490 value
1	Negative control	0.008
2		0.008
3		0.098
MV		0.038
4	Positive control	0.662	0.624
5		1.220	1.182
6		2.260	2.222
MV		1.381	1.343
7	Test item	0.160	0.122
8		0.190	0.152
9		0.157	0.119
MV		0.169	0.131

 Table 3: in vitro irritation score. MV= mean value.

Cornea Nr.	Test item	Corrected opacity	Corrected OD490 value	IVIS
1	Negative control	0.23	0.008	1.49
2		1.74	0.008
3		0.80	0.098
MV		0.92	0.038
4	Positive control	121.86	0.624	133.42
5		116.25	1.182
6		101.73	2.222
MV		113.28	1.343
7	Test item	9.27	0.122	9.49
8		5.26	0.152
9		8.04	0.119
MV		7.52	0.131

 Table 4: Historical mean in vitro irritation score of the positive control

	IVIS Positive Control
Mean value (MV)	125.20
Standard deviation (SD)	17.75
MV – 2xSD	89.71
MV + 2xSD	160.70
Number of replicates providing historical mean: 27

 Table 5: Historical mean in vitro irritation score of the negative control

	IVIS Positive Control
Mean value (MV)	1.23
Standard deviation (SD)	0.79
MV – 2xSD	-0.35
MV + 2xSD	2.80
Number of replicates providing historical mean: 27

 

Interpretation of results:

other: Expert judgement: not Category 1

Conclusions:

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay. The following mean in vitro irritation score was calculated: 9.49. No prediction can be made regarding the classification of the test substance to the evaluation criteria. For safety reasons, the test substance is classified as Category 2.

Executive summary:

In a primary ex vivo eye irritation guideline study (bovine corneal opacity and permeability assay) under GLP conditions,750 mg of neat potassium hexafluorophosphate was applied on corneas for 4 hours. Irritation was scored by the method recommended in guideline OECD 437.

In this study, the mean in vitro irritation score was 9.49. According to GHS, no prediction can be made using this value. For precautionary measures, potassium hexafluorophosphate is classified voluntarily as mildly irritating to eyes (GHS Category 2).

Additional information

The potential of potassium hexafluorophosphate to induce skin corrosion or irritation was assessed using a validated in vitro model (RhE). Two guideline studies according to OECD 431 were conducted which differed only in the application of the test material: neat or moistened with 25 µL water, respectively. Neither neat nor moistened potassium hexafluorophosphate showed any corrosive properties in the in vitro skin corrosion assay.

In consequence, a study according to OECD guideline 439 was carried out where potassium hexafluorophosphate was moistened with 25 µL water and applied to the in vitro skin model (RhE). Potassium hexafluorophosphate showed no irritant effects in this assay.

In a primary ex vivo eye irritation study (BCOP) according to OECD guideline 437, neat potassium hexafluorophosphate was applied on corneas for 4 hours. The mean in vitro irritation score was 9.49. According to UN GHS, no prediction can be made using this value. For precautionary measures, potassium hexafluorophosphate is classified voluntarily as mildly irritating to eyes (UN GHS Category 2B).

All controls confirmed the validity of these studies.

Justification for classification or non-classification

In a primary ex vivo eye irritation study, application of neat potassium hexafluorophosphate resulted in a mean in vitro irritation score was 9.49. According to UN GHS, no prediction can be made using this value. For precautionary measures, potassium hexafluorophosphate is classified voluntarily as mildly irritating to eyes (UN GHS Category 2B).