Dioctyl phosphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From July 24th to 27th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 28th, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl phosphonate

EC Number:

217-315-6

EC Name:

Dioctyl phosphonate

Cas Number:

1809-14-9

Molecular formula:

C16H35O3P

IUPAC Name:

dioctyl phosphonate

Test material form:

liquid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: Commercially available EpiOcular kit. The EpiOcular tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm^2.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control (sterile demineralised water) and positive control (methyl acetate).

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µl for each tissue.

Duration of treatment / exposure:

28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80-100 % relative humidity.

Duration of post- treatment incubation (in vitro):

120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80-100 % relative humidity.

Number of animals or in vitro replicates:

Two replicates

Details on study design:

- Details of the test procedure used:
GENERAL PRINCIPLE OF THE STUDY:
Eye irritant materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential.

PRE-TESTS:
-ASSESSMENT OF DIRECT REDUCTION of MTT
The test item was tested for the ability of direct MTT reduction. To test for this ability, 50 µl of the liquid test item were added to 1 ml of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 ml of MTT reagent plus 50 µl of H2O demin. was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
- ASSESSMENT OF COLOURED OR STAINING TEST ITEM
The test item is colourless. To assess whether the test item will become coloured after contact with water or isopropanol, 50 µl of the liquid test item were added to 1 ml of sterile H2O demin. in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour minutes. After incubation, no colour development was visible. Therefore, the main test was per-formed without colourant controls.

MAIN TEST:
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37 ± 1°C.
6-well-plates were labelled with test item, negative control and positive control and filled with 1 ml assay medium. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 ml fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours 30 minutes.
After overnight incubation, the tissues were pre-wetted with 20 µl DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 µl of the controls and the test item were applied in duplicate in 1 minute intervals. This was done in such a fashion that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of the exposure time, the inserts were removed from the plates in 1 minute intervals using sterile forceps and rinsed immediately with DPBS. Then, the tissues were immediately transferred to and immersed in 5 ml of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature.
After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 ml assay medium. For post-treatment incubation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

MTT ASSAY AND EXTRACTION
A 24-well-plate was prepared with 300 µl freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into the empty 24-well-plate. Into each well, 2 ml isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was sealed and stored in the refrigerator overnight. On the next day, the plate was shaken for 2 hours at room temperature, protected from light.

MEASURAMENT
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µl solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µl isopropanol were also pipetted. The plate was read in a plate spectrophotometer at 570 nm.

EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

CALCULATION
% VIABILITY= [OD corrected test item/OD corrected mean negative control]*100%

- RhCE tissue construct used, including batch number: the EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm^2. EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Myln-ské Nivy 73, 82105 Bratislava, Slovakia (Batch number 23797).

- Doses of test chemical used: 50 µl

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80-100 % relative humidity
Post-exposure incubation: 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80-100 % relative humidity.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): not applicable: the pre-tests regarding direct reduction of MTT and regarding colouring potential of the test item were negative.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): two replicates

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): quantification of MTT formazan performed at 570 nm.

- Description of the method used to quantify MTT formazan: spectrophotometer.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
% Viability > 60 % : Non eye irritant: No GHS category for eye irritation
% Viability: ≤ 60 %: Eye irritant: GHS category 1 or 2

- ACCEPTANCE CRITERIA:
OD of negative control: > 0.8 and < 2.5
Tissue viability of positive control: < 50 % (relative to negative control)
Variation within two tissue replicates of test item and controls: < 20 %

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

Mean of 2 tissue replicates

Value:

ca. 10.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS OF MAIN TEST:
After treatment with the test item, the mean value of relative tissue viability was reduced to 10.7 %.

RESULTS OF PRELIMINARY TESTS:
In the preliminary test to assess the direct reduction of MTT, MTT reagent did not change its colour after incubation with test item; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
In the preliminary test to assess coloured test item, no colour development was visible; therefore, the main test was performed without colourant controls.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean viability for negative and positive controls was in the range of historical data.

ACCEPTANCE OF RESULTS:
OD of negative control: > 0.8 and < 2.5: fulfilled (1.5)
Tissue viability of positive control: < 50% (relative to negative control): fulfilled (34.1 %)
Variation within two tissue replicates of test item and controls: < 20%: fulfilled (3.2 % for negative control; 4.4 % for positive control and 1.5 % for test item).

Any other information on results incl. tables

Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Dioctyl phosphonate-
Tissue 1	1	1.584	0.593	0.211
	2	1.588	0.588	0.210
Tissue 2	1	1.552	0.524	0.189
	2	1.523	0.523	0.185

 Mean Absorbance Negative Control, Positive Control and Test Item (Note Mean Blank = 0.036)

Designation	Negative Control	Positive Control	Dioctyl phosphonate-
Mean – blank (Tissue 1)	1.550	0.555	0.175
Mean – blank (Tissue 2)	1.502	0.488	0.151

% Viability Positive Control and Test Item

Designation	Positive Control	Dioctyl phosphonate-
% Viability (Tissue 1)	36.3 %	11.4 %
% Viability (Tissue 2)	31.9 %	9.9 %
% Viability Mean	34.1 %	10.7 %

Historical data

Parameter	Optical Density Negative Control	Relative Viability % Positive Control
	Demineralised H2O	Methyl acetate
Exposition time	28 minutes
Mean	1.851	31.3 %
Standard deviation	0.283	8.2 %
Range	1.253 – 2.437	12.4 – 57.2 %
Study17021405G891	1.526	34.1%

Applicant's summary and conclusion

Interpretation of results:

other: eye irritant according to the CLP Regulation (EC n.1272/2008)

Conclusions:

Dioctyl phosphonate is eye irritant according to the CLP Regulation (EC n.1272/2008).

Executive summary:

The potential eye irritation of Dioctyl phosphonate was examined according to OECD TG 492 (EpiOcular^TMEye Irritation test).

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes. 50 µl of the liquid test were applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: after treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.5. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 34.1 % (< 50 %).

Variation within tissue replicates was acceptable (< 20 %).

After treatment with the test item, the mean value of relative tissue viability was 10.7 %.

This value is well below the threshold for eye irritation potential (≤ 60 %).

According to the OECD TG 492, the EpiOcular^TMEye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).

Considering the results of in vitro tests for skin and eye irritation, the structure of the molecule and its pH in water (3.9), Dioctyl phosphonate is classified H319 (eye irritant category 2) according to the CLP Regulation (EC.n.1272/2008).