N-benzoyl-5'-O-(p,p'-dimethoxytrityl)-2'-deoxycytidine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-09 to 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

adopted in 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-benzoyl-5'-O-(p,p'-dimethoxytrityl)-2'-deoxycytidine

EC Number:

266-605-9

EC Name:

N-benzoyl-5'-O-(p,p'-dimethoxytrityl)-2'-deoxycytidine

Cas Number:

67219-55-0

Molecular formula:

C37H35N3O7

IUPAC Name:

N-{1-[(2R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxyoxolan-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES:
- Source: Spear Products Inc., 520 Springfield Street, Coopersburg, PA 18036. Fresh eyes were collected from adult cows roughly between the ages of 12-30 months at a slaughterhouse and transported in Hanks Balanced Salt Solution (HBSS) with Ca++ and Mg++ (containing penicillin/streptomycin) on ice to PSL. Corneas were dissected and the assay begun as soon as possible after delivery.

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20%

Duration of treatment / exposure:

4 hours at 32 °C

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were carefully examined for defects (opacity, scratches, pigmentation, etc.) shortly after arrival to the facility. Any eyes with defects were omitted from the study and discarded. The tissue surrounding the eye ball was carefully pulled away, and the cornea was excised, leaving a rim of sclera approximately 2 to 3 mm wide. The isolated corneas were maintained in HBSS until all were dissected. Prior to mounting, corneas were again visually examined for blemishes. All corneas free from defects were then mounted in corneal holders (BASF, Duratec) with the endothelial side against the O-ring of the posterior chamber. Each holder was uniquely identified by a matching number on both the anterior and posterior chambers. The anterior chamber was then positioned on top of the cornea and tightened with screws to hold the cornea securely in place and completely seal the chambers. Both chambers were then filled, starting with the posterior chamber to help maintain the normal shape of the cornea, with pre-warmed cMEM, ensuring that no bubbles were formed. The corneas were incubated for a minimum of one hour at ~32 °C to allow them to equilibrate with the medium.

INITIAL OPACITY AND CORNEA SELECTION
At the end of the initial incubation period, the medium in both chambers was replaced with fresh pre-warmed cMEM. An initial opacity (illuminance) measurement (I) was performed on each cornea using an opacitometer (Model: BASF-OP3.0, Duratec). Only corneas having an initial illuminance reading I > Io/1.1651 lux (927 lux) were used for the assay. Io is the empirically determined illuminance through cornea holders containing cMEM without a cornea. Three corneas with an initial opacity closest to the median opacity of all usable corneas were selected as the negative control corneas. Remaining corneas were randomly assigned to test and positive control groups. The medium was removed from the anterior chamber just prior to treatment with test or control substance.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period, the test or control substance was removed from the anterior chamber and the epithelium was washed at least three times with MEM containing phenol red until no colour change occurred. The corneas were given a final rinse with cMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
Corneal opacity:
The following formula was used to calculate the opacity using the initial and final illuminance measurements taken from each cornea. The values for a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity= (Io/I) - b)/a
Where: a= 0.0251, b= 0.9894, Io= 1080
The value Io is the illuminance through medium without a cornea. This value was previously determined using the mean illuminance through cornea holders containing cMEM. This value is re-evaluated periodically by the testing facility. I is the initial illuminance being evaluated.

Change in Opacity:
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity from the final opacity. The average change in opacity was calculated for the negative control corneas and used to calculate the corrected opacity.
Corrected Opacity:
The corrected opacity value for each cornea treated with test substance or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each treated and positive control cornea. The mean corrected opacity value of each treatment group was then calculated by averaging the corrected opacity values.

Permeability (UV/VIS Spectrophotometer Method):
After the final opacity measurement, the medium was removed from the anterior chamber of the holder. One milliliter of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber of all holders. After addition of the sodium fluorescein solution, all corneas were incubated in a horizontal position for approximately 90 minutes at ~32 °C. Once incubation was complete, the medium from the posterior chamber of each holder was transferred to individual, pre-labeled sample tubes. Aliquots, measuring 200 µL, of each sample were added onto a 96-well plate, in duplicate, with two additional wells filled with cMEM (blanks). The optical density at a wavelength of 490 nm was then measured using a spectrophotometer.
Corrected OD490:
The mean OD490 obtained for the blank wells was calculated. The mean blank OD490 was subtracted from the OD490 of each test or positive control well (corrected OD490).
Final Corrected OD490:
The final-corrected OD490 of the test substance(s) and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea. The mean final corrected OD490 for each treatment group was calculated.
Final-corrected OD490 = corrected OD490 treated cornea – average-corrected negative control OD490

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Corrected opacity and permeability values calculated above were used for each group to determine the IVIS for each treatment group using the following formula:
IVIS= mean corrected opacity value + (15 x mean corrected OD490 value)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

11

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

Proper conduct of the BCOP was confirmed via a positive response with imidazole and a negative response with 0.9% NaCl. For detailed results please refer to table 2 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 2: Classification summary

Group	Treatment	Physical State	RESULTS OF LABORATORY TESTING
			Avg Corrected Opacity	Avg Corrected OD	IVIS[1]	Category
						UN GHS	EPA
1	Negative Control (0.9% NaCl)	Liquid	-	-	-	-	-
2	Positive Control (Imidazole)	Powder	100.8	2.508	138	Category 1	I
3	Test Substance  (N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)- 2'-deoxycytidine)	Powder	10.7	0.007	11	No prediction can be made	III

[1] In Vitro Irritancy Score (IVIS)= mean corrected opacity value + (15 x mean corrected OD₄₉₀value)

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, based on the mean in vitro irritation score of 11 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), no prediction can be made regarding the classification of the test substance.

Executive summary:

The eye irritation potential of N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine (99.65% purity) was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item was suspended in water to gain a 20% concentration. A mean in vitro irritation score of 11 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no prediction can be made regarding the classification of the substance.