Phosphinylidynetrimethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 2020-10-19 - 2020-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

OECD Guideline for the Testing of Chemicals, Part 492, adopted 18. Jun. 2019, “Reconstructed Human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye Irritation or serious eye damage”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphinylidynetrimethanol

EC Number:

213-924-6

EC Name:

Phosphinylidynetrimethanol

Cas Number:

1067-12-5

Molecular formula:

C3H9O4P

IUPAC Name:

[bis(hydroxymethyl)phosphoryl]methanol

impurity 1

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

H2O4S.2Na

IUPAC Name:

sodium sulphate

impurity 2

Reference substance name:

Sodium formate

EC Number:

205-488-0

EC Name:

Sodium formate

Cas Number:

141-53-7

Molecular formula:

CHO2Na

IUPAC Name:

sodium formate

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid: viscous

Details on test material:

- Appearance: Light yellow to colourless ropy liquid.
- Storage conditions: Room temperature (approx. 20 °C), protected from light.
- Batch No.: 20200616
- Production date: 2020-06-16.
- Expiry date: 2021-06-16.

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemi-cals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcularTM EIT can be used as a replacement of the in vivo Draize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Specification:
Commercially available EpiOcularTM kit.
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 cm2.
Origin:
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 20. Oct. 2020
Batch no.: 30681
The cells used to produce EpiOcular tissues are screened for potential biological contaminants. None of the following potential contaminants were detected:
HIV-1 virus (Oligonucleotide-directed amplification)
Hepatitis B virus (Oligonucleotide-directed amplification)
Hepatitis C virus (Oligonucleotide-directed amplification)
Bacteria, yeast, other fungi (long-term antibiotic, antimycotic free culture)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL / tissue
- Concentration (if solution): undiluted

VEHICLE
none

Duration of treatment / exposure:

28 minutes

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number
Commercially available EpiOcularTM kit.
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 cm2.
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 20. Oct. 2020
Batch no.: 30681
- Doses of test chemical and control substances used
Test chemical: 50 µg/tissue
Controls: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and the test item were applied in duplicate in one-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 ≥ 95% relative humidity.
At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed 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 incu-bation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1% CO2 and ≥ 95% relative humidity.
After the post-treatment incubation, the MTT assay was performed.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Spectrophotometer, 570 nm
- Description of the method used to quantify MTT formazan
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 ≥ 95% relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
Measurement
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 pipetted also. The plate was read in a plate spectrophotometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
OD value; > 60% of control
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Parameter Optical Density Negative Control Relative Tissue Viability Positive Control
Demineralised H2O Methyl acetate
Exposure time 30 minutes 30 minutes
Mean 1.888 32.6%
Standard deviation 0.260 7.2%
Range 1.167 - 2.437 12 - 57%
Current study 1.932 29.1%

- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
The validity of the EpiOcularTM test at the laboratory was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

- Positive and negative control means and acceptance ranges based on historical data
Yes
- Acceptable variability between tissue replicates for positive and negative controls
< 20%
- Acceptable variability between tissue replicates for the test chemical
< 20%

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: % tissue viability

Run / experiment:

mean test item

Value:

90.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: % tissue viability

Run / experiment:

mean positive control

Value:

29.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not different

Any other information on results incl. tables

1) Findings and Results

a) Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table: Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.033	0.033	0.033	0.034	0.033	0.034	0.034	0.035	0.034

 

The absorbance values of negative control, test item and positive control are given in the following table:

 

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

Designation	Measurement	Negative Control	Positive Control	Test item
Tissue 1	1	1.925	0.610	1.801
	2	1.892	0.600	1.794
Tissue 2	1	2.018	0.586	1.784
	2	2.027	0.586	1.778

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in the table below (= corrected values).

 

Table: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Test item
Mean – blank (Tissue 1)	1.875	0.571	1.764
Mean – blank (Tissue 2)	1.989	0.552	1.747

 

b) Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

 

Table: % Viability Positive Control and Test Item

Designation	Positive Control	Test item
% Viability (Tissue 1)	29.6%	91.3%
% Viability (Tissue 2)	28.6%	90.4%
% Viability Mean	29.1%	90.9%

 

c) Assessment

Eye hazard potential is assessed using the criteria given in the following table:

 

Table: Assessment of Eye Hazard Potential

% Viability	Assessment	UN GHS classification
> 60 %	Non eye irritant	No Category
≤ 60 %	At least eye irritant	No prediction can be made (category 1 or 2)

 

d) Validity

Validity criteria and results are stated in the following table:

 

Table: Validity

Criterion	Demanded	Found
Mean OD of negative control	> 0.8 and < 2.8	1.9
% mean relative viability of positive control	< 50% of negative control	29.1%
Variation within replicates	< 20%	5.9% (negative control) 1.0% (positive control) 0.9% (test item)

Values for negative control and for positive control were within the range of historical data of the test facility.

Therefore, the experiment is considered valid.

 

2) Discussion

One valid experiment was performed. Under the conditions of the test,THPO, Tris(hydroxymethyl)phosphine oxideis considered non-eye irritant in the EpiOcular^TMEye Irritation Test.

After treatment with the test item, the mean value of relative tissue viability was reduced to 90.9%. This value is below the threshold for eye irritation potential (≤ 60%).

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.9 (> 0.8 and < 2.8).

The positive control induced a decrease in tissue viability as compared to the negative control to 29.1%.

The variation within the replicates of the controls and the test item was acceptable (< 20%).

For these reasons, the result of the test is considered valid.

Applicant's summary and conclusion

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

- mean value of relative tissue viability was 90.9 %
- test item is considered non-eye irritant under the conditions of the test [since the value is well above the threshold for eye irritation potential (≤ 60%)]

Executive summary:

The study was conducted under GLP according to OECD guideline 492 on the registration substance Tris(hydroxymethyl)phosphine oxide (THPO). The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation / corrosion potential of the test substance to the eye in vitro.

One valid experiment was performed. The test item THPO 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 item was applied to two tissue replicates.

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 < 2.8, OD was 1.9. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 29.1% (< 50%).

The variation within tissue replicates of the controls and the test item was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 90.9%.

This value is above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

Under the conditions of the test, THPO is considered non-eye irritant in the EpiOcular^TMEye Irritation Test.