Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July to 5 October 2018
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
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiDerm Skin Model has been validated for corrosivity testing in an international validation study and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model, EPI-200, MatTek Corporation, Ashland MA, U.S.A.
- Tissue batch number(s): Lot no.: 28864

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0ºC.
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0ºC.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 4 tissues for the test tem together with a negative control and positive control. Two tissues were used for a 3-minute exposure and two tissues for a 1-hour exposure.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue viability obtained after 3 minutes exposure is less than 50% compared to the negative control tissues 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 relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

VEHICLE: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL 8N KOH
Duration of treatment / exposure:
3 minute and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
4

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute application
Value:
88
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour application
Value:
18
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Colour interference with MTT: Alkenyl phosphonate was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that Tributyl(ethyl) phosphonium diethylphosphate did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Tributyl(ethyl) phosphonium diethylphosphate is not corrosive to the skin in the EpiDerm Skin Model
Executive summary:

An in vitro skin corrosion test of Tributyl(ethyl) phosphonium diethylphosphate was performed in a human three dimensional epidermal model (EpiDerm) according to the OECD No. 431 guideline and GLP. The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. The test item was applied undiluted (50 μL) directly on top of the skin tissue.

Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

The positive control had a mean relative tissue viability of 6.3% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 13%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 88% and 18%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.