Registration Dossier

Administrative data

Description of key information

Not irritant for the skin
Slightly irritant for the eyes

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17th January 2013 to 11th of March
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, well conducted on the similar substance Reaction mass of dipotassium phosphonate and Phosphonic acid, potassium salt (1:1). Rationale for Read Across is attached at point 13
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 761/2009 amending Regulation (EC) No. 440/2008, B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test
GLP compliance:
yes (incl. certificate)
Species:
other: in vitro
Details on test animals and environmental conditions:
In vitro test
Type of coverage:
other: in vitro
Amount / concentration applied:
20 μL/well (see table 1)
Duration of treatment / exposure:
15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature.
Treatment was carried out staggering samples of approximately 1 minute.
Positive control: an intermediate re-spreading was carried out approximately after 7 minutes of incubation.
A 42 hour recovery period
Number of animals:
In vitro
Details on study design:
The test system EPISKIN™ is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.
The principle of the RhE test method is based on the premise that chemicals are able to penetrate the stratum corneum and irritant chemicals are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS RN 298-93-1] into a blue formazan salt that is quantitatively measured after extraction from tissues. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels.

Test System: EPISKIN™
Commercial Name: EPISKINTM - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
Batch: 13-EKIN-008
Arrived at RTC: 05 March 2013
Functional controls: Quality controls: histology scoring, magnitude of viability and barrier function (IC50 determination).
Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.
A certificate of analysis can be found in Addendum III.

Preparation of the Test System
Examination at arrival
Temperature indicator: pale grey (suitable for use)
pH indicator: orange (suitable for use)
Placing in culture: according to the supplier procedure, the test system was shipped on Monday and received on Tuesday. At arrival, the plate was openedunder a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate (supplied) in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium.
Pre-treatment incubation period: culture dishes were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

Media
SkinEthic Maintenance Medium: SkinEthic; batch: 13-MAIN3-011
SkinEthic Assay Medium: SkinEthic; batch: 13-ESSC-002 and 13-ESSC-009
Dulbecco’s Phosphate buffered saline (D-PBS): GIBCO; batch: 1098754
Sterile water: Baxter; batch: 12D1810
MTT Reagent:Sigma; batch: MKBB1495
MTT Stock Solution: MTT Reagent 3 mg/mL in D-PBS
Stored at ambient conditions, protected from light; used on the day of dilution
MTT Ready-to-use Solution: MTT Stock Solution diluted 1:10 (v/v) with SkinEthic Assay Medium (final concentration 0.3 mg/mL of MTT)
Stored at ambient conditions, protected from light. Used within 1 hour from preparation
Acidic Isopropanol
(0.04 N HCl in isopropanol): 12 N HCl (Fluka; batch: 0001434790) added to
2-propanol (BDH; batch: 09D170512)
Stored at ambient conditions
Used within 1 month from preparation

Experimental procedure
Preliminary test
Direct MTT reduction test (Step 1) : Non-specific reduction of MTT was evaluated as follows: two mL of MTT Ready-to-use Solution was incubated with 20 µL of test item at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.
Colouring potential test (Step 2): Chemicals’ colouring potential was assessed for potential interaction with the test system.
10 μL of the test item was added to 90 µL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes.
Colouring of the solution at the end of the incubation time was evaluated.

Main Assay
Treatment: A Main Assay was carried out including the test item, positive and negative controls. Sample Test System Treatment are reported in table 1.
Washing: At the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert.
The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.
Post-exposure period: A 42 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.
Media collection for further analysis: At the end of the incubation period, the plates were shaked on a plate shaker for about 15 minutes at approximately 300 rpm/min.
A volume of 1.6 mL of each incubation medium was removed and stored at 20°C for possible future analysis.These samples were not analysed.

MTT Assay: The tissue insert and controls were incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hours at 37°C, 5% CO2 and saturated humidity.
At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 μL of acidic isopropanol.
Tubes were mixed by vortexing and preserved for approximately 3 days at 4°C to allow formazan extraction.
At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 μL) of acidic isopropanol were analysed and used as blank.



Irritation / corrosion parameter:
other:
Value:
> 92.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3h. Max. score: 100.0. Reversibility: other: Not applicable. Remarks: A value > 50 indicates no irritation potential. (migrated information)
Irritant / corrosive response data:
Preliminary study
Results of this preliminary test can be found in Table 2.
Before the main irritation study, a preliminary test was carried out to assay the compatibility of the test item with the test system.
In a first step, the test item was assayed for the ability of reducing MTT per se. No interaction was recorded between the test item and MTT in the test conditions. Thus no additional controls were added in the main phase for the evaluation of MTT non specific reduction. No precipitate was noted.
In a second step, the test item was assayed for the ability of colouring water per se. No colouring potential of the test item in contact with water was recorded. For this reason, no additional controls were added in the main phase for the evaluation of non specific colouring potential.

Main irritation study
Raw data and data elaboration of the main irritation study are reported in Table 3.
The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.
Positive control results indicated an appropriate cell death with an acceptable relative cell viability (4.3% of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 0.5).
Based on the stated criteria: mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.
The test item did not induce any relevant reduction of MTT staining in any replicates. The mean cell viability was 92.1% when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 5.2 lower than 18).

Table 2. Preliminary test

Direct MTT reduction test (Step 1)
Test item (mL) MTT ready to use solution (mL) Container Incubation condition Colour Observation
20 2.0 well 3 h at 37°C, 100% nominal humidity, 5% CO2 Yellowish (no interaction)
Colouring potential test (Step 2)
Test item (mL) MTT ready to use solution (mL) Container Incubation condition Colour Observation
10 90 Eppendorf tube 15’, ambient condition, in agitation Colourless solution

Table 3. Main test

Test Viability%
Blank (negative control) 100
Positive control 4.3
Test Item A 92.1
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The potential of the test item Potassium phosphonate KH2PO3/KH2PO3 to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKINTM. Based on the mean cell viability (92.1%) when compared to the negative control, the test item should be considered not irritant. The negative and positive controls gave the expected results and the study was accepted as valid.
Executive summary:

The potential of the test item Potassium phosphonate KH2PO3/KHPO3 to be irritant to the skin was investigated through anin vitroskin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKINTM. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439.

The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period.The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and colouring waterper se.

No interaction was recorded between the test item and MTT in test conditions similar to those of the Main Assay. Moreover, no colouring potential of the test item in contact with water was recorded. Thus, no additional control was added in the main phase for the evaluation of non specific colour generation which may influence evaluation of results.

In the Main Assay,the test item was applied as supplied in three replicates at the treatment level of 20mL/epidermisunit, each measuring 0.38 cm2(treatment level: 53mL/cm2).

Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20mL/epidermisunit.

The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.       

The positive control caused the expected cell death (4.3% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.5).

Based on the stated criteria: mean viability ≤ 40% and SD of % viability ≤ 18, the assay was regarded as valid.       

The test item did not induce cell death in any replicate with a mean cell viability of 92.1% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 5.2 (lower than 18).

Based on the results obtained, the test itemPotassium phosphonate – Multicomponentis classified as not irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
other: in vitro MTT test
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013, March
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, well conducted on the similar substance Reaction mass of dipotassium phosphonate and Phosphonic acid, potassium salt (1:1). Rationale for Read Across is attached at point 13
Qualifier:
according to
Guideline:
other: MTT test method
Deviations:
no
Principles of method if other than guideline:
The method allows to evaluate a toxic event and related cytotoxicity by a colorimetric assay. Solution of MTT in balanced saline is yellowish in color. Mitochondrial dehydrogenase of viable cells cleaves the tetrazolium ring yielding blue/purple MTT crystals which are insoluble in aqueous solutions. Crystals formed by viable cells are retained in the polycarbonate filter used as substrate for the 3D construct. Intense purple colour of the cultures indicates the viability of the cell at the basal layer, whereas colour remains white when necrosis occurs. Negative controls are of a dark blue colour and positive controls are white-yellow.
MTT crystals are extracted by isopropanol and optical density is measured at 570 nm.
GLP compliance:
yes (incl. certificate)
Species:
other: HCE corneal epithelium model
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source:SKINETHIC LABORATORIES
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL

- Concentration (if solution):The test item was tested at the dose defined for liquid (30 µL).

Duration of treatment / exposure:
1 hour
Observation period (in vivo):
not required
Number of animals or in vitro replicates:
no aminals required
Details on study design:
The test was performed on triplicate tissues for MTT and in simplicate for histological analysis.
The positive control was Ethanol (99,96%) and as negative control saline solution (NaCl 0,9%) was used. 30 µL of controls (liquids) were directly and uniformly applied topically.

SCORING SYSTEM:cell viability

OTHER: the test was performed in triplicate
Irritation parameter:
other: Cell viability
Basis:
mean
Time point:
other: 1h + 16h
Score:
ca. 47
Max. score:
100
Reversibility:
not specified
Remarks on result:
other: SD ± 0.039
Irritant / corrosive response data:
The prediction model for eye irritation ciassification is based on cell viability (MTT-modified protocol) after 1h+16h exposure (SOP M 04).
A cut-off of 50% is accepted for discriminating between potential irritant (<50 %) and not classified (>50 %). Positive control (Ethanol) satisfies acceptance criteria if it is classified as irritant (<50 %).
Mean tissue viability is <50 % Irritant (I) R36
Mean tissue viability is >50 % Not Classified (NC)
Interpretation of results:
slightly irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A cell viability > 50% corresponding to the cut-off value for eye irritant classification has been quantified for the test item (0,47%).
Executive summary:

The present study has been conducted in order to assess in vitro, on a Human Corneal Epithelium model (HCE), the eye irritation potential of Potassium Phosphonate KH2PO3/K2HPO3

Eye irritation potential of the test item was assessed at 1h followed by product washing and a post incubation period of 16h by using the MTT test method to quantify the residual cell viability. A complementary histo-morphological analysis was associated.

According to the adopted prediction model based on MTT results the chemicals have been classified as reported in the following table.

Classification based on cell viability-MTT results.

CHEMICAL 1h+16h
KH2PO3 IR
ETOH IR
  IR

(NC=Not Classified; IR=Irritant for the eye, R 36 )

The scoring of the complementary histo-morphological analysis was in agreement with the cell viability results.

A cell viability < 50% corresponding to the cut-off value for eye irritant classification has been quantified for the test item (0,47%).

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of the test item Potassium phosphonate KH2PO3/KHPO3 to be irritant to the skin was investigated through an in vitro skin irritation study (Bisini, 2013) using a commercial reconstructed human epidermis (RhE) model named EPISKINTM. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439.

The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period.The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

No interaction was recorded between the test item and MTT in test conditions similar to those of the Main Assay. Moreover, no colouring potential of the test item in contact with water was recorded.

The test item did not induce cell death in any replicate with a mean cell viability of 92.1% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 5.2 (lower than 18).

Based on the results obtained, the test itemPotassium phosphonate – Multicomponentis classified as not irritant to the skin.

No test for corrosion has been performed, since the substance is not irritant for the skin.

According to ECHA progress report 2010 (p. 32), it is accepted that in vitro methods for skin irritation represent a full replacement of the in vivo method in a tiered testing strategy and in conjunction with in vitro skin corrosivity tests, if necessary. A positive result in the human skin model for irritation does not need to be confirmed by additional testing and shall lead to classification.

A study has been conducted in order to assess in vitro, on a Human Corneal Epithelium model (HCE), the eye irritation potential ofPotassium Phosphonate KH2PO3/K2HPO3 (De Servi, 2013)

Eye irritation potential of the test item was assessed at 1h followed by product washing and a post incubation period of 16h by using the MTT test method to quantify the residual cell viability. A complementary histo-morphological analysis was associated.

According to the adopted prediction model based on MTT results the substance has been classified as eye irritant.

A second eye irritation/corrosion study (Longobardi C., 2013) has been presented on the monocomponent substance. This form exists at lower pH (< 5.5) with a higher potential for irritation property, therefore the proposed eye classification deriving from the HCE test is confirmed


Justification for selection of eye irritation endpoint:
The study is more sensitive for slightly irritating substances and contributes then better to the classsification of the substance

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

Skin irritation/corrosion:

The substance is not classified an skin irritant because in the selected test it doesn't meet the classification criteria of the CLP regulation n. 1272/2008.

Eye irritation:

The substance is classified a eye irritant based on HCE in vitro test