Tricobalt bis(orthophosphate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-01-26 to 2022-04-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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:

adopted 18 June 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tricobalt bis(orthophosphate)

EC Number:

236-655-6

EC Name:

Tricobalt bis(orthophosphate)

Cas Number:

13455-36-2

Molecular formula:

Co3(PO4)2

IUPAC Name:

tricobalt bis(orthophosphate)

impurity 1

Reference substance name:

Dicobalt diphosphate

EC Number:

238-689-7

EC Name:

Dicobalt diphosphate

Cas Number:

14640-56-3

Molecular formula:

Co2P2O7

IUPAC Name:

dicobalt diphosphate

impurity 2

Reference substance name:

cobalt phosphate octahydrate

Cas Number:

10294-50-5

Molecular formula:

Co3(PO4)2.8H2O

IUPAC Name:

cobalt phosphate octahydrate

Test material form:

solid: particulate/powder

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

Pre-treatment:
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 7951221). The tissues were incubated at standard culture conditions for 30 minutes.
Treatment:
The test item was applied as supplied, for 5 hours and 56 minutes at standard culture conditions, at the dose of 50 mg to the entire surface of 2 living and 2 killed RhCE tissue replicates.
In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 211021) were carried out. The controls were applied, as supplied, at the dose of 50 μL, to the surface of 2 RhCE tissue replicates during 5 hours and 58 minutes at standard culture conditions.

Duration of post- treatment incubation (in vitro):

Post-exposure incubation period:
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 7951221). The rinsed tissues were checked for any coloration and noted to be white. Purple residual test item was noted on the epidermis after the rinse.
This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for a 18 hours and 02 minutes post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

Number of animals or in vitro replicates:

2

Details on study design:

Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 μL of a MTT solution at 1.0 mg/mL for 2 hours and 46 minutes at standard culture conditions. The NSC living and NSC killed control tissues were incubated in assay medium (MatTek Corporation, batch No. 111521ISA) instead of MTT solution in order to generate NSC controls.
The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol for 2 hours and 20 minutes at room temperature in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).
The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Remarks:

mean

Run / experiment:

3rd run

Value:

69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

corrected value (NSMTT)

Results 2

Irritation parameter:

other: mean optical density

Run / experiment:

3rd run

Value:

0.682

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptability criteria:
- Tissues treated with the positive control substance should show a mean tissue viability < 50%.
- The difference of viability between two tissue replicates should be less than 20%.
- Negative control: OD values of the two replicates should be in the range > 0.8 and < 2.8.
As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control.

Any other information on results incl. tables

The results were expressed as a viability percentage compared with the negative control:

Viability % = Mean OD test item x 100/Mean OD negative control

As the test item was identified as producing potential MTT reduction, true tissue viability is calculated as follows:

True viability % = (Mean OD test item – Mean OD NSMTT)x 100/Mean OD negative control

Evaluation and interpretation of the results

The OD values obtained with the replicate tissue extracts for each test item were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results are interpreted as follows:

The test item is identified as not requiring classification and labelling according to UN GHS No Category:

➢ if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

In this case, no further testing in other test methods is required.

The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1):

➢ if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%.

When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 69.0%, versus 35.8% in the positive control (Methyl acetate).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions and in accordance with the Regulation EC No. 1272/2008, the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category. No hazard statement and the signal word are required.