Trimethyl phosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethyl phosphate

EC Number:

208-144-8

EC Name:

Trimethyl phosphate

Cas Number:

512-56-1

Molecular formula:

C3H9O4P

IUPAC Name:

trimethyl phosphate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No. of test material: Sigma Aldrich, Saint Louis, USA; MKBX2207V
- Expiration date of the batch: 26/06/2022
- Purity test date: 97 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: yes

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult human-derived epidermal keratinocytes; three dimensional human epidermis model

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), EPISKIN SNC Lyon, France
- Tissue batch number(s): 17-EKIN-032
- Expiry date: 14 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsed thoroughly with approximately 25 mL PBS 1x solution to remove the test item from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL. The obtained solution was used within one hour; it was protected from light before used.
- Incubation time: After the exposure of test item was terminated by rinsing with PBS, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, ≥95% humidified atmosphere.
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

A volume of 50 μL test item was applied evenly to the epidermal surface of the two test skin units respectively. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

4h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

88

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

101

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (1-2)

Value:

95

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Positive and negative controls showed the expected cell viability values within acceptable limits.
All assay acceptance criteria were met, the experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Trimethyl phosphate – 97% can be classified as non-corrosive.

Executive summary:

EpiSkinTM SM test of the test item Trimethyl phosphate – 97% has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 29 July 2016.

Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5 % CO2 in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively.

For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 95 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure.

Positive and negative controls showed the expected cell viability values within acceptable limits.

All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions. In conclusion, the test item Trimethyl phosphate – 97% can be classified as non-corrosive.