Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Aug 2017 to 01 Oct 2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Lot number: 2000850599
Analysis date: 19 August 2016
Specific details on test material used for the study:
Test item information
Identification: Tri-o-tolylphoshpine
Appearance: Off-white crystalline powder (determined by Charles River Den Bosch)
Batch: B64002 (Pfizer Lot no. 2000850599)
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature
Stable under storage conditions until: Unknown

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, U.S.A.
Source strain:
not specified
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE : EpiDerm Skin Model (EPI-200, Lot no.: 26787 kit C+D). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm2) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Source
MatTek Corporation, Ashland MA, U.S.A.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C

Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
-After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 µl Milli-Q water as a negative control were added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
- Spectrophotometer: 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Barrier function: ET-SO assay. 100 ul 1% Triton X-100, 4 time-points
- Contamination: Long term antibiotic and antimycotic free culture. Acceptability criteria- no contamination. Result was sterile.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin [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.
Control samples:
other: Positive Control: Potassium hydroxide, Negative Control: Milli-Q water
Amount/concentration applied:
The skin was moistened with 25 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 30.5 to 36.2 mg of the solid test item was added into the 6-well plates on top of the skin tissues.

For the negative and positive controls, 2 tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues were treated with 50 µl 8N KOH (positive control) for both the 3-minute and 1-hour time point.
Duration of treatment / exposure:
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
Number of replicates:
2 replicates for the 3-minute exposure and 2 replicated for the 1-hour exposure.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: Mean OD570
Remarks:
3 minute application
Run / experiment:
Test Item
Value:
1.838
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Irritation / corrosion parameter:
other: Mean OD570
Remarks:
1 hour application
Run / experiment:
Test item
Value:
1.666
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Direct-MTT reduction: The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere 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:
In conclusion, Tri-o-tolylphoshpine is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate Tri-o-tolylphoshpine for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch B64002 (Pfizer Lot no. 2000850599) of the test item was an off-white crystalline powder. Skin tissue was moistened with 25 µl of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 8.4% 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 (lower acceptance limit ≥0.8 and upper acceptance limit < or = 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < or = 10%, 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 112% and 94%, 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.

In conclusion, Tri-o-tolylphoshpine is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.