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EC number: 241-143-0 | CAS number: 17084-13-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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 %. - 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”.
- 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 %. - 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”.
- 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 %. - 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”.
Referenceopen allclose all
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 |
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 |
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 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- 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”. - 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).
Reference
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 |
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).
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