Reaction mass of 604-916-7 and (4R,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2),

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2011 to 12 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 437: "Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants" (adopted September 07, 2009).

Deviations:

no

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)

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ". Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OTWG

Version / remarks:

The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: INVITTOX 127

Version / remarks:

In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Fundam Appl Toxicol 18:442-449

Version / remarks:

Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(4S,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2)

EC Number:

604-916-7

Cas Number:

153621-80-8

Molecular formula:

C23H30N4O17S2

IUPAC Name:

(4S,4aS,5aR,12aS)- 4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide sulfate (1:2)

Test material form:

solid: particulate/powder

Details on test material:

Purity 80-100%

Test animals / tissue source

Species:

cattle

Strain:

other: an isolated bovine cornea

Details on test animals or tissues and environmental conditions:

Bovine eyes were used as soon as possible after slaughter on the same day.

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% (w/w) test substance solution

Duration of treatment / exposure:

240 +/- 10 minutes

Observation period (in vivo):

Immediate opacity measurement and permeability evaluation of the cornea

Number of animals or in vitro replicates:

Three corneas for each treatment group (total 9 corneas)

Details on study design:

6.4.1. 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 1 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 1 C. The corneas were incubated for the minimum of 1 hour at 32 1 C.

6.5. 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 treatmentgroup.

6.6. Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 l 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 10 minutes at 32 1 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.

6.7. 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.

6.8. 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 5 minutes at 32 1 C.

6.9. 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 l 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.

6.10. Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Multiskan spectrum version 1.00 (Thermo labsystems, Breda, The Netherlands) for optical density measurement.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

96

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

0.144

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The negative control responses of the opacity and permeability values were less than the upper limits
of the laboratory historical range indicating that the negative control did not induce irritancy on the
corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 95 and
within the historical positive control data range (APPENDIX 3, Table 6). It was therefore concluded
that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

9-Nitrominocycline induced severe ocular irritation through one endpoint (opacity) mainly, resulting in a mean in vitro irritancy score of 98 after 240 minutes of treatment.
Since 9-Nitrominocycline induced an IVIS ≥ 55.1, it is concluded that 9-Nitrominocycline is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.