Phosphinylidynetrimethanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 2018-03-13 - 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphinylidynetrimethanol

EC Number:

213-924-6

EC Name:

Phosphinylidynetrimethanol

Cas Number:

1067-12-5

Molecular formula:

C3H9O4P

IUPAC Name:

[bis(hydroxymethyl)phosphoryl]methanol

Test material form:

liquid: viscous

Details on test material:

yellowish liquid, viscous, turbid
CAS no.: 1067-12-5
Storage conditions: at +10 to +25°C, in the tightly closed original container and stored in a cool, dry and well-ventilated place, protected from heat and sunlight

Specific details on test material used for the study:

Tris(hydroxymethyl)phosphine oxide (THPO) was applied as liquid test item topically undiluted to the model skin surface.

In vitro test system

Test system:

human skin model

Remarks:

The EpiDermTM model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by
diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-[4,5-
Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue), into a blue formazan salt that was quantitatively measured after extraction from tissues.
Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS Category 1 or Category 2).
The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200, Lot no. 25899) MatTek
- Tissue batch number(s): 00267
- Quality analysis date: 2018-04-25

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 35 min., follwed by room temperture for 25 min.
- Temperature of post-treatment incubation (if applicable): 42 hour, temperature not specified

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 2 measurements

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant or corrosive to skin if the viability is less or equal than 50%
- The test substance is considered to be non-corrosive to skin if the viability is greater than 50%

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): D-PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 , Mean viability (n=3), THPO

Value:

>= 54.7 - <= 61.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

mean viability: 57.8 %

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control (vehicle): yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table 'any other information on materials and methods incl. tables'

Any other information on results incl. tables

Table 1 Summarized Results of in vitro Skin Irritation

	Optical density [OD540] mean tissue (n = 3)	Mean Viability (n=3) [%]	CV [%]
D-PBS (negative control)	1.320	100.0	5.2
DL-alpha-alanine	1.217	92.2	5.6
5 % SDS (positive control)	0.099	7.5	9.3

CV : coefficient of variation

SDS : sodium dodecyl sulphate

D-PBS : Dulbecco's phosphate buffered saline

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Remarks:

not irritant to skin

Conclusions:

- mean value of relative tissue viability was reduced to 57.8%
- test item is considered non-irritant to skin under the conditions of the test [since obtained value is above the threshold for skin irritation potential (50%)]

Executive summary:

The purpose of this study was to determine cytotoxic properties of Tris(hydroxymethyl)phosphine oxide (THPO) to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin according to OECD Guideline 439 and in compliance with GLP criteria.The EpiDerm^TM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. THPO was applied to the model skin surface; D-PBS was used as the negative control and 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to THPO was 57.8 % of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. THPO was considered to be non-cytotoxic and predicted to be non-irritant to skin.

All acceptance criteria required were fulfilled.

Under the present test conditions, THPO was non-cytotoxic and predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin. Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category).