Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: test conducted under GLP conditions and acc. to OECD guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

EC Number:

246-129-8

EC Name:

Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

Cas Number:

24292-60-2

Molecular formula:

C21H28N7O17P3.2Na

IUPAC Name:

Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

Details on test material:

- Physical state: white powder
- Storage condition of test material: in refrigerator (2-8°C) in the dark

Test system

Vehicle:

physiological saline

Controls:

not required

Amount / concentration applied:

20% (w/w) solution (750 µl)

Duration of treatment / exposure:

240 minutes of treatment

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such
as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored at 32°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The
Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) 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 °C. The corneas were incubated for the minimum of 1 hour at 32°C.

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 Ul of the negative control, 20% (w/v) Imidazole solution (positive control) or 20% (w/w) test substance solution were introduced onto the epithelium of the cornea. 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 minutes at 32 °C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The
numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg
Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 minutes at 32 °C.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled
according to holder number. 360 Ul of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.


The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

opacity

Run / experiment:

1

Value:

5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

Permeability

Value:

-0.004

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The corneas treated with b-NADP, Disodium salt showed opacity values ranging from 4 to 6 and the permeability value was -0.004 for all three corneas. The corneas were clear after the 240 minutes of treatment with b-NADP, Disodium salt. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 3.9 to 5.9 after 240 minutes of treatment with b-NADP, Disodium salt.

Any other information on results incl. tables

Summary of opacity, permeability andin vitro scores

Treatment	Mean Opacity	Mean Permeability	MeanIn vitroIrritation Score^1, 2
Negative control	0	0.000	0
Positive control	72	2.076	103
b-NADP, Disodium salt	5	-0.004	4.9

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

it is concluded that this test is valid and that b-NADP, Disodium salt is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

Screening for the eye irritancy potential of b-NADP, Disodium salt using the Bovine Corneal Opacity and Permeability test (BCOP test). The possible ocular irritancy of b-NADP, Disodium salt was tested through topical application for 240 ± 10 minutes.

 

The study procedures described in this report were based on the most recent OECD and EC guideline. The test substance was applied as a 20% (w/w) solution (750 µl) directly on top of the corneas.

 

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitroirritancy score of the positive control (20% (w/v) Imidazole) was 103 and within the historical positive controldata range.It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

The mean in vitro irritancy score was 4.9 after 240 minutes of treatment with b-NADP, Disodium salt.

Since the mean in vitro irritancy score for b-NADP, Disodium salt was below 55.1 after 240 minutes treatment b-NADP, Disodium salt is considered to be not severe irritant or corrosive.

 

Finally, it is concluded that this test is valid and that b-NADP, Disodium salt is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.