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Description of key information

Tributyl(ethyl) phosphonium diethylphosphate appeared to be non corrosive to the skin, while it induced serious eye damange in the Bovine Corneal Opacity and Permeability test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 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
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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 19 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: in-vitro test
Strain:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
TEST MATERIAL
- Source: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Acclimation period: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. Then, the corneas were dissected and incubated in incubation medium at 32 +/- 1°C in an incubation chamber for 1 hour.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 +/- 1°C
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
An amount of 750 µl of either the negative control, positive control or test item was applied directly on the corneas in such a way that the cornea was completely covered.
Duration of treatment / exposure:
The corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1°C.
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes at 32+/-1°C.
Details on study design:
The aim of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea.

PROTOCOL
- For each treatment group (negative control, positive control and test item), three replicates were used.
- The opacity of each cornea was determined before exposure.
- The corneas were exposed for 10 minutes.
- After the incubation the change in opacity for each individual cornea (including the negative control) was determined.
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.

INTERPRETATION
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro irritancy score (IVIS)
- A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritant.
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:
in vitro irritation score
Value:
59
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test substance showed opacity values ranging from 30 to 34 and permeability values ranging from 1.482 to 2.287. The corneas were turbid after the 10 minutes of treatment with Tributyl(ethyl) phosphonium diethylphosphate. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, the in vitro irritancy scores ranged from 56 to 64 after 10 minutes of treatment with Tributyl(ethyl) phosphonium diethylphosphate.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (Ethanol) was 47 and was within two standard deviations of the current historical positive control mean.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Tributyl(ethyl) phosphonium diethylphosphate induced serious eye damage in the Bovine Corneal Opacity and Permeability test.
Executive summary:

A GLP compliant in-vitro study was performed according to OECD Guideline 437 in order to evaluate the ocular irritation properties of Tributyl(ethyl) phosphonium diethylphosphate on an isolated bovine cornea. The possible ocular irritancy of the test item was tested through topical application for approximately 10 minutes. For each treatment group (negative control, positive control and test item), three replicates were used. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. After treatment, the IVIS (in vitro irritancy score) in the controls is within the acceptability criterion. The positive control showed clear irritating effects. Tributyl(ethyl) phosphonium diethylphosphate induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 59 after 10 minutes of treatment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion/irritation:

Based on the results of the in vitro EpiDerm Skin Model test, Tributyl(ethyl) phosphonium diethylphosphate is non-corrosive to skin. Taking into account the experimental results, a classification as irritating to skin is proposed based on precautionary principle.

Eye irritation:

A GLP compliant in-vitro study according to OECD Guideline 437 was performed in order to evaluate the ocular irritation properties of Tributyl(ethyl) phosphonium diethylphosphate on isolated bovine cornea. Since Tributyl(ethyl) phosphonium diethylphosphate induced an IVIS > 55, it is concluded that the substance induced serious eye damage in the Bovine Corneal Opacity and Permeability test.

Justification for classification or non-classification

Based on the experimental data, Tributyl(ethyl) phosphonium diethylphosphate is classified as Cat 2; H315 "irritating to skin" and Cat. 1; H318 'Causes serious eye damage' in accordance with the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.