2,4(1H,3H)-Pyrimidinedione, 1-(3,5-di-O-acetyl-2-deoxy-.beta.-L-erythro-pentofuranosyl)-5-methyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[(2S,3R,5S)-3-acetoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl]methyl acetate

EC Number:

610-122-1

Cas Number:

433733-94-9

Molecular formula:

C14H18N2O7

IUPAC Name:

[(2S,3R,5S)-3-acetoxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl]methyl acetate

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes were used as soon as possible after slaughter.
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

306.5 to 353.1 mg

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

3 Negative control
3 Positive control
3 Test item

Details on study design:

The medium from the anterior compartment was removed and 750 ul of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Diacetyl Thymidine (LDT600-C3) was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (306.5 to 353.1 mg).
The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity 1

Value:

-1.4

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base.

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity 2

Value:

-1.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity 3

Value:

2.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

irritancy score 1

Value:

-0.4

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

in vitro irritation score

Run / experiment:

irritancy score 2

Value:

-0.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

in vitro irritation score

Run / experiment:

irritancy score 3

Value:

3.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Diacetyl Thymidine (LDT600-C3) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.