Tri-2-propyn-1-yl phosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tris(prop-2-yn-1-yl) phosphate

EC Number:

853-966-8

Cas Number:

1779-34-6

Molecular formula:

C9H9O4P

IUPAC Name:

tris(prop-2-yn-1-yl) phosphate

Specific details on test material used for the study:

Name Phosphoric acid,tri-2-propargyl ester (LDY196)
Batch no. H219121101
CAS no. 1779-34-6
Composition Phosphoric acid,tri-2-propargyl ester 99 %
Storage room temperature (20 ± 5 °C)
Stability stable under storage conditions
Appearance Yellow liquid
Purity ≥ 99 % LDY196
Homogeneity homogeneous

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old.

Test system

Controls:

yes

Amount / concentration applied:

The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.

Duration of treatment / exposure:

within 1 hour

Details on study design:

PERFORMANCE OF THE STUDY
Preparation of Test System
After having carefully cleaned and sterilized the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used.
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. The formation of bubbles was prevented.
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the cornea holder in an opacitometer and recording the illuminance (unit: LUX).
None of the corneas showed an opacity greater than seven opacity units; therefore, all corneas were used.

Description of the Method
Preparations
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
Application
For each treatment group (negative control solution, test item or positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL of negative control solution, 750 µL of test item or 750 µL of positive control were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the controls and the test item, the following treatment procedure was performed:
Closed Chamber Method:
The respective substance (negative control solution, test item or positive control) was ap-plied by pipetting 750 µL of the appropriate liquid through the refill hole in the anterior holder on the cornea. The controls and the test item were given on the epithelium that the cornea was evenly covered.
Exposure time of the controls and test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing the anterior chambers with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chambers were filled with cMEM without phe-nol red and the cornea holders were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time the final opacity value of each cornea was recorded. 
Permeability Test
After the recording of the final opacity values, the cMEM without phenol red was removed from both chambers of each cornea holder. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM. Then 1 mL sodium fluores-cein solution was added to the front chamber of each cornea holder for the detection of permeability of the corneas.
For the controls and the test item a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a horizontal position. After incubation, the content of each posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 1.16

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this test, the test item Phosphoric acid,tri-2-propargyl ester (LDY196) showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 1.16.
According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.