Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicon orthophosphate
EC Number:
234-858-4
EC Name:
Silicon orthophosphate
Cas Number:
12037-47-7
Molecular formula:
O16P4Si3
IUPAC Name:
silicon(4+) tetraphosphate
Details on test material:
- Name of test material (as cited in study report): silicon orthophosphate
- Physical state: solid, light grey
- Analytical purity: >80% (w/w)
- Batch No.: 5
- Storage condition of test material: at room temperature, dry atmosphere, protected from light

Test animals / tissue source

Species:
other: isolated bovine corneas
Strain:
not specified

Test system

Vehicle:
physiological saline
Controls:
other: number of eyes for the test item: 3
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL test item preparation
Duration of treatment / exposure:
4 hours ± 5 minutes at 32 ± 1 °C
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
Fresh eyes from the slautherhouse were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in the corneal holders, they were carefully examined for defects and any defective eyes were discarded. The chambers were filled with RPMI containing 1% FBS and 2 mM L-glutamine and incubated for one hour at 32 ± 1°C in a water bath. After the equilibration period, the medium was removed from the chambers and replaced by fresh medium. An initial opacity measurement was performed on each of the corneas using the calibrated opacitometer. Afterwards the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed and the epithelium was washed three times with medium. Once the medium was free of test substance, the cornea was fully rinsed with complete RPMI and opacity measurement was performed. Afterwards, the medium was removed from the chambers and filled with fresh medium. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1°C. Then the medium was removed and its optical density at 490 nm was determined, using a spectrophotometer. 3 corneas each were used for the test item, as negative control treated with physiological saline 0.9% NaCl or as positive control treated with imidazole 20% in physiological saline 0.9% NaCl.

The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article 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: final-corrected OD490 = (OD490 - mean blank OD490) - average-corrected negative control OD490. The mean OD490 value of each treatment group was calculated by averaging the final corrected OD 490 values of the treated corneas for that treatment condition. The following formula was used to determine the in vitro irritation score (IVIS): IVIS= mean opacity value + (15x mean OD490 value). Evaluation of the mean scores as follows: 0-3 = non irritant, 3.1-25 = mild irritant, 25.1 – 55 = moderate irritant, 55.1 – 80 = severe irritant, >80.1 = very severe irritant.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: opacity
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
93.33
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: permeability
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
-0.014
Reversibility:
other: not applicable
Remarks on result:
other: Test substance
Irritation parameter:
other: IVIS
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
93.13
Reversibility:
other: not applicable
Remarks on result:
other: Test Substance
Irritation parameter:
other: opacity
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
164.67
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: permeability
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
1.966
Reversibility:
other: not applicable
Remarks on result:
other: Positive control
Irritation parameter:
other: IVIS
Basis:
other: mean out of all 3 eyes
Time point:
other: 4 h
Score:
194.16
Reversibility:
other: not applicable
Remarks on result:
other: Positive control

Any other information on results incl. tables

Table 1: Opacity data

Cornea No.

Test item

Initial Opacity

Final Opacity

Change of Opacity Value

Corrected Oapcity Value

1

Negative Control

3

3

0

 

2

3

4

1

 

3

3

3

0

 

Mean Value

3.00

3.33

0.33

 

4

Positive Control

3

199

196

195.67

5

3

150

147

146.67

6

3

155

152

151.67

Mean Value

3.00

168.00

165.00

164.67

7

Test Item

2

102

100

99.67

8

2

102

100

99.67

9

2

83

81

80.67

Mean Value

2.00

95.67

93.67

93.33

Table 2: Permeability data

Cornea No.

Test Item

OD490

Corrected OD490 Value

1

Negative Control

0.026

 

2

0.010

 

3

0.011

 

Mean Value

0.016

 

4

Positive Control

1.996

1.980

5

1.870

1.854

6

2.079

2.063

Mean Value

1.981

1.966

7

Test Item

0.003

-0.013

8

0.002

-0.014

9

0.001

-0.015

Mean Value

0.002

-0.014

Table 3: In vitro irritation score

Cornea No.

Test Item

Corrected Opacity Value

Corrected OD490 Value

IVIS

1

Negative Control

0.00

0.026

 

2

1.0

0.010

 

3

0.00

0.011

 

Mean Value

0.33

0.016

0.57

4

Positive Control

195.67

1.980

 

5

146.67

1.854

 

6

151.67

2.063

 

Mean Value

164.67

1.966

194.16

7

Test Item

99.67

-0.013

 

8

99.67

-0.014

 

9

80.67

-0.015

 

Mean Value

93.33

-0.014

93.13

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: Cat. 1, H318
DSD: Xi, R41