Dioctyl phosphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 16th to June 19th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28th, 2015

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl phosphonate

EC Number:

217-315-6

EC Name:

Dioctyl phosphonate

Cas Number:

1809-14-9

Molecular formula:

C16H35O3P

IUPAC Name:

dioctyl phosphonate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors.

Details on animal used as source of test system:

SOURCE ANIMAL
- Source:Human adult donors.

Justification for test system used:

Test system used according to the OECD TG 439. 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.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM – 0.38 cm2
- Supplier: SkinEthic Laboratories (4, A. Flemig – 69366 Lyon – France)
- Tissue batch number(s): 17-EKIN-024 (alive tissues)
- Shipping date: Monday 12 June 2017
- Delivery date: Tuesday 13 June 2017. Note: at arrival the test system was examinated and it resulted suitable for use (temperature indicator:pale grey; pH indicator: orange). Plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 ml/well SkinEthic Maintenance Medium. Culture plates were placed in the incubator at 37 °C, 5 % CO2 and saturated humidity for approximately 24 hours.

PRELIMINARY TESTS:
Before the Main Assay, a preliminary tests were carried out to evaluate the compatibility of the test item with the test system.
- Direct-MTT reduction: 2 ml of MTT ready-to-use solution (0.3 mg/ml) was incubated with 20 μl of test item at 37 °C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.
- Colour interference with MTT: 20 μl of the test item was added to 180 µl of distilled water in at transparent tube and the resulting suspension mixed by using a vortex for 15 minutes. Colouring of the suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm.

MAIN TEST
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 ± 1 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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: not observed

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml/well of MTT ready-to-use solution
- Incubation time: 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 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 (14000 rpm for 2 minutes) and aliquots of 200 µl from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200µl) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range , an MTT formazan calibration curve was performed.
- Wavelength: the absorbance was evaluated at 595 nm.

NUMBER OF REPLICATE TISSUES: 3 replicates for negative control, for positive control and for test item and two replicates for test item without MTT. Note:the test item without MTT is Non Specific Colouring potential (NSC living) using to evaluate the potential colouring of the test item.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive and non-irritant to skin if the viability after an exposure period of 15 minutes followed by 42 ± 1 hour recovery period is greater than 50%.

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
– Blank controls: mean OD value < 0.1.
– Negative controls: mean OD value ≥ 0.6 and ≤ 1.5, SD of % viability ≤ 18.
– Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and SD of % viability ≤ 18.
– Test item: SD of % viability ≤ 18.

Control samples:

other: Positive control: 5% (w/v) sodium dodecyl sulphate (SDS) solution; negative control: Dulbecco’s phosphate buffered saline (D-PBS); Test item without MTT (to evaluate the potential interferring ability of the test item on Optical Density).

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 μl/epidermis unit of liquid test item for each replicate (total number of replicates: 3).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 20 µl/epidermis unit of Dulbecco’s phosphate buffered saline (D-PBS) (total number of replicates: 3).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 20 µl/epidermis unit of 5%(w/v) sodium dodecyl sulphate (SDS) solution for each replicate (total number of replicates: 3).
- Concentration (if solution): 5 %(w/v) sodium dodecyl sulphate (SDS) solution, obtained by 1:1 dilution in sterile water of a sterile commercial 10 %(w/v) SDS solution in water.

NSC living (Test item without MTT to evaluate the potential colouring interaction of the test item)
- Amount(s) applied (volume or weight with unit): 20 μl/epidermis unit of liquid test item without MTT for each replicate (total number of replicates: 2).

Duration of treatment / exposure:

15 ± 0.5 minutes in a ventilated cabinet at room temperature. At the end of the exposure, each tissue was rinsed with approximately 25 ml of sterile D-PBS, filling and empting the tissue insert.

Duration of post-treatment incubation (if applicable):

42 ± 1 hour by incubation at 37 °C, 5% CO2 and saturated humidity.

Number of replicates:

- Test item, negative control, positive control: three replicates each;
- Test item without MTT (NSC living): two replicates. Note:The test item without MTT is Non Specific Colouring potential (NSC living) using to evaluate the potential colouring of the test item.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates (A1, A2, A3) for test item (for each replicate the reading of absorbance was performed in duplicate)

Value:

ca. 53

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:

DETAILS ON RESULTS:
The mean cell viability of the test item treated tissues, after the blank subtraction, was 53 % when compared to the negative control

- OTHER EFFECTS:
- Direct-MTT reduction: no interaction of test item with MTT. Before the main assay a preliminary test was performed to assay the ability of test item to reduce MTT per se. Two ml of MTT ready-to-use solution (0.3 mg/ml) was incubated with 20 μl of test item at 37 °C, 5 % CO2 and saturated humidity for 3 hours. At the end of the incubation period with the MTT, yellow drops in suspension were noted. Since no colour change was observed, it was concluded that the test item could not interact with MTT.
- Colour interference with MTT: possible colour interference of test item. Before the main assay a preliminary test was performed to assay the ability of test item of colouring the water per se. 20 μl of the test item was added to 180 μl of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes.Blank suspension was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 1.396, indicating that the test item has a potential interfering ability. Based on the results obtained, additional controls were added in the Main Assay for the evaluation of Non Specific Colouring potential (NSCliving). Colour interference (NSC) was 1 %, thus only the blank subtraction was performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank controls: the mean Optical Density of Blank Controls was 0.036, lower than the maximum acceptable value (0.1).
- Acceptance criteria met for negative control: the negative control gave the expected baseline value (Mean of Optical Density values of the three replicates= 0.6 and it is inside the range of acceptability: mean OD value ≥ 0.6 and ≤ 1.5) and viability (SD of % viability of three replicates : 3.6 % lower than the maximum acceptable value 18 %).
- Acceptance criteria met for positive control: the positive control caused the expected cell death (Mean of cell viability of the three replicates = 6% - lower than the maximum acceptable value 40%) and variability (SD of % viability for three replicates equal to 2.1 % - lower than the maximum acceptable value 18%).
- Acceptance criteria met for test item : the mean cell viability of the test item treated tissues after the blank subtraction was 53 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.1 % - lower than the maximum acceptable value (18%).

Applicant's summary and conclusion

Interpretation of results:

other: not classified as skin irritant, according to the CLP Regulation (EC n.1272/2008)

Conclusions:

Mean cell viability of the test item treated tissues = 53 % (after blank subtraction).
The test item Dioctyl phosphonate is identified as not irritant to the skin according to the CLP Regulation (EC n.1272/2008).

Executive summary:

The potential of the test item Dioctyl phosphonate to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD TG 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 ± 1 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 a first step, the test item was assayed for the ability of reducing MTT per se. At the end of the incubation period with the MTT, no colour change was noted indicating that the test item could not interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A blank suspension was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 1.396, indicating that the test item has a potential interfering ability. Based on these results, additional controls were added in the Main Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 μl/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 μl/cm^2). 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 20 μl/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control.

In the Main Assay, the negative control gave the expected baseline value (mean Optical Density values equal or 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 negative control 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 (6 % of cell viability when compared to the negative control) and variability (SD of % viability equal to 2.1). Based on the stated criteria (mean viability ≤ 40 % and SD of % viability ≤ 18), the assay was regarded as valid. The colouring interference (NSC) was 1 %; thus only the black subtraction was performed. The mean cell viability of the test item treated tissues, after the blank subtraction, was 53 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.1 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item Dioctyl phosphonate is classified as not irritant to the skin.